Methods of treating cancer using pd-1 axis binding antagonists and mek inhibitors

ABSTRACT

The present invention describes combination treatment comprising a PD-1 axis binding antagonist and a MEK inhibitor and methods for use thereof, including methods of treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. provisionalapplication Ser. No. 61/574,406, filed Aug. 1, 2011, the contents ofwhich are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The provision of two distinct signals to T-cells is a widely acceptedmodel for lymphocyte activation of resting T lymphocytes byantigen-presenting cells (APCs). Lafferty et al, Aust. J. Exp. Biol.Med. ScL 53: 27-42 (1975). This model further provides for thediscrimination of self from non-self and immune tolerance. Bretscher etal, Science 169: 1042-1049 (1970); Bretscher, P. A., P.N.A.S. USA 96:185-190 (1999); Jenkins et al, J. Exp. Med. 165: 302-319 (1987). Theprimary signal, or antigen specific signal, is transduced through theT-cell receptor (TCR) following recognition of foreign antigen peptidepresented in the context of the major histocompatibility-complex (MHC).The second or co-stimulatory signal is delivered to T-cells byco-stimulatory molecules expressed on antigen-presenting cells (APCs),and induce T-cells to promote clonal expansion, cytokine secretion andeffector function. Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). Inthe absence of co-stimulation, T-cells can become refractory to antigenstimulation, do not mount an effective immune response, and further mayresult in exhaustion or tolerance to foreign antigens.

In the two-signal model T-cells receive both positive and negativesecondary co-stimulatory signals. The regulation of such positive andnegative signals is critical to maximize the host's protective immuneresponses, while maintaining immune tolerance and preventingautoimmunity. Negative secondary signals seem necessary for induction ofT-cell tolerance, while positive signals promote T-cell activation.While the simple two-signal model still provides a valid explanation fornaive lymphocytes, a host's immune response is a dynamic process, andco-stimulatory signals can also be provided to antigen-exposed T-cells.The mechanism of co-stimulation is of therapeutic interest because themanipulation of co-stimulatory signals has shown to provide a means toeither enhance or terminate cell-based immune response. Recently, it hasbeen discovered that T cell dysfunction or anergy occurs concurrentlywith an induced and sustained expression of the inhibitory receptor,programmed death 1 polypeptide (PD-1). As a result, therapeutictargeting of PD-1 and other molecules which signal through interactionswith PD-1, such as programmed death ligand 1 (PD-L1) and programmeddeath ligand 2 (PD-L2) are an area of intense interest.

PD-L1 is overexpressed in many cancers and is often associated with poorprognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, the majority oftumor infiltrating T lymphocytes predominantly express PD-1, in contrastto T lymphocytes in normal tissues and peripheral blood T lymphocytesindicating that up-regulation of PD-1 on tumor-reactive T cells cancontribute to impaired antitumor immune responses (Blood 2009114(8):1537). This may be due to exploitation of PD-L1 signalingmediated by PD-L1 expressing tumor cells interacting with PD-1expressing T cells to result in attenuation of T cell activation andevasion of immune surveillance (Sharpe et al., Nat Rev 2002) (Keir M Eet al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of thePD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing oftumors.

The inhibition of PD-1 axis signaling through its direct ligands (e.g.,PD-L1, PD-L2) has been proposed as a means to enhance T cell immunityfor the treatment of cancer (e.g., tumor immunity). Moreover, similarenhancements to T cell immunity have been observed by inhibiting thebinding of PD-L1 to the binding partner B7-1. Furthermore, combininginhibition of PD-1 signaling with other signaling pathways (e.g. MAPKpathway, “MEK”) that are deregulated in tumor cells may further enhancetreatment efficacy. However, an optimal therapeutic treatment wouldcombine blockade of PD-1 receptor/ligand interaction with an agent thatdirectly inhibited tumor growth, optionally further including uniqueimmune enhancing properties not provided by PD-1 blockade alone. Thereremains a need for such an optimal therapy for treating, stabilizing,preventing, and/or delaying development of various cancers.

All references, publications, and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention describes a combination treatment comprising a MEKinhibitor (which has direct tumor targeted effects and immune enhancingproperties) and a PD-1 axis binding antagonist.

Provided herein are methods for treating cancer or slowing progressionof cancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and a MEK inhibitor.

Also provided herein is use of a PD-1 axis binding antagonist in themanufacture of a medicament for treating or delaying progression ofcancer in an individual in combination with a MEK inhibitor. Alsoprovided herein is use of a MEK inhibitor in the manufacture of amedicament for treating or delaying progression of cancer in anindividual in combination with a PD-1 axis binding antagonist. Alsoprovided herein is use of a PD-1 axis binding antagonist and a MEKinhibitor in the manufacture of medicaments for treating or delayingprogression of cancer in an individual. Also provided herein is amanufacturing process of medicaments for treating or delayingprogression of cancer in an individual, characterized by the use of aPD-1 axis binding antagonist and a MEK inhibitor. Also provided hereinis a PD-1 axis binding antagonist for use in combination with a MEKinhibitor for treating or delaying progression of cancer in theindividual. Also provided herein is a MEK inhibitor for use incombination with a PD-1 axis binding antagonist for treating or delayingprogression of cancer in the individual.

The cancer treated may contain a BRAF V600E mutation, a BRAF wildtype, aKRAS wildtype, or an activating KRAS mutation. The cancer may be amelanoma, a colorectal cancer, a non-small cell lung cancer, an ovariancancer, a breast cancer, a prostate cancer, a pancreatic cancer,hematological malignancy or a renal cell carcinoma. The cancer may be atearly stage or at late stage. In some embodiments, the individualtreated is a human.

In some embodiments, the treatment results in sustained response in theindividual after cessation of the treatment. In some embodiments, thetreatment produces a complete response, a partial response, or stabledisease in the individual.

Also provided herein are methods of enhancing immune function in anindividual having cancer comprising administering an effective amount ofa PD-1 axis binding antagonist and a MEK inhibitor. In some embodiments,the individual is a human.

Also provided herein is use of a PD-1 axis binding antagonist in themanufacture of a medicament for enhancing immune function in anindividual having cancer in combination with a MEK inhibitor. Alsoprovided herein is use of a MEK inhibitor in the manufacture of amedicament for enhancing immune function in an individual having cancerin combination with a PD-1 axis binding antagonist. Also provided hereinis use of a PD-1 axis binding antagonist and a MEK inhibitor in themanufacture of medicaments for enhancing immune function in theindividual having cancer. Also provided herein is a manufacturingprocess of medicaments for enhancing immune function in an individual,characterized by the use of a PD-1 axis binding antagonist and a MEKinhibitor. Also provided herein is a PD-1 axis binding antagonist foruse in combination with a MEK inhibitor for enhancing immune function inthe individual having cancer. Also provided herein is a MEK inhibitorfor use in combination with a PD-1 axis binding antagonist for enhancingimmune function in the individual having cancer. In some embodiments,the individual is a human.

In some embodiments, the PD-1 axis binding antagonist is a PD-1 bindingantagonist, a PD-L1 binding antagonist or a PD-L2 binding antagonist. Insome embodiments, the PD-1 binding antagonist inhibits binding of PD-1to PD-L1 and/or binding of PD-1 to PD-L2. In some embodiments, the PD-1binding antagonist is an antibody (e.g., antibody MDX-1106, CT-011 andMerck 3745 described herein), an antigen binding fragments thereof, animmunoadhesin, a fusion protein, or an oligopeptide. In someembodiments, the PD-1 binding antagonist is an immunoadhesin comprisinga PD-L2 extracellular domain fused to a Fc domain (e.g., AMP-224described herein). In some embodiments, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or binding of PD-L1 to B7-1. Insome embodiments, the PD-L1 binding antagonist is an antibody (e.g.,antibody YW243.55.S70, MPDL3280A and MDX-1105 described herein), anantigen binding fragments thereof, an immunoadhesin, a fusion protein,or an oligopeptide. In some embodiments, the PD-L2 binding antagonistinhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2binding antagonist is an antibody, an antigen binding fragments thereof,an immunoadhesin, a fusion protein, or an oligopeptide.

In some embodiments, the MEK inhibitor is a compound of the formula (I),(II), (III), (IV), (V), or (VI) as described here below, or apharmaceutically acceptable salt or solvate thereof.

In some embodiments, the MEK inhibitor is a competitive inhibitor ofMEK. In some embodiments, the MEK inhibitor is more selective againstactivating KRAS mutation. In some embodiments, the MEK inhibitor is anallosteric inhibitor of MEK. In some embodiments, the MEK inhibitor ismore selective against an activating BRAF mutation. In some embodiments,the MEK inhibitor is selected from the group consisting of G02442104,G-38963, G02443714, G00039805, and GDC-0973, or a pharmaceuticallyacceptable salt or solvate thereof.

In some embodiments, the MEK inhibitor is administered continuously orintermittently. In some embodiments, the MEK inhibitor is administeredbefore administration of the PD-1 axis binding antagonist,simultaneously with administration of the PD-1 axis binding antagonist,or after administration of the PD-1 axis binding antagonist. In someembodiments, the MEK inhibitor and the PD-1 axis binding antagonist areadministered with different dosing frequency.

In another aspect, provided is a kit comprising a PD-1 axis bindingantagonist and/or a MEK inhibitor for treating or delaying progressionof a cancer in an individual or enhancing immune function in anindividual having cancer. The kit may comprise a PD-1 axis bindingantagonist and a package insert comprising instructions for using thePD-1 axis binding antagonist in combination with a MEK inhibitor totreat or delay progression of cancer in an individual, or enhancingimmune function in an individual having cancer. The kit may comprise aMEK inhibitor and a package insert comprising instructions for using theMEK inhibitor in combination with a PD-1 axis binding antagonist totreat or delay progression of cancer in an individual, or to enhanceimmune function in an individual having cancer. The kit may comprise aPD-1 axis binding antagonist and a MEK inhibitor, and a package insertcomprising instructions for using the PD-1 axis binding antagonist andthe MEK inhibitor to treat or delay progression of cancer in anindividual, or to enhance immune function in an individual havingcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows enhanced MHC I surface expression on melanoma andcolorectal tumor cell lines upon treatment with MEK inhibitor. (A)Histogram showing increased MHC I expression on the surface of humantumor cell lines treated with MEK inhibitor. (B) Histogram showingincreased MHC I expression on the surface of mouse tumor cell linestreated with MEK inhibitor.

FIG. 2 is a histogram showing that treatment of human melanoma celllines (5/8 cell lines of which were BRAF mutant; BRAF wild-type cellsindicated with asterisk) with BRAF inhibitor did not upregulate MHC Isurface expression.

FIG. 3 shows that treatment of human peripheral blood mononuclear cellswith MEK inhibitor did not upregulate MHC I surface expression. (A-D)Histogram showing unaltered MHC I surface expression in CD4+ T cells,CD8+ T cells, B cells, or monocytes upon MEK inhibitor treatment.

FIG. 4 demonstrates that co-stimulatory signals make T cells responsivedespite MEK inhibitor treatment. (A) Graph of CD8⁺ T cells levels showsthat MEK inhibitor treatment reduced T cell proliferation and activationnormally induced by stimulation of CD3. (B) Graph of CD8⁺ T cells showthat co-stimulation of CD3 and CD28 was sufficient to overcome theinhibitory effect of MEK inhibitor treatment.

FIG. 5 shows that MEK inhibitor treatment enhanced maturation andactivation of dendritic cells stimulated with anti-CD40 antibodies.(A-C) Histogram showing dendritic cells stimulated with anti-CD40antibodies and treated with MEK or BRAF inhibitor. MEK inhibitorenhanced DC activation as evidenced by upregulation of DC surfaceactivation markers CD83, MHC II and CD86. (D-F) Graphs of activateddendritic cell levels demonstrates that MEK inhibitor enhanced DCactivation in a dose dependent manner.

FIG. 6 is a graph showing reduced serum levels of immunosuppressive andpro-tumor cytokines in in vivo models of cancer. (A and C)Immunosuppressive cytokine IL-10 was decreased 7 days followingco-treatment with anti-PD-L1 antibodies and MEK inhibitor as compared totreatment with anti-PD-L1 or MEK inhibitor treatment alone. (B and D)The pro-tumor chemokine KC was decreased upon co-treatment withanti-PD-L1 antibodies and MEK inhibitor as compared to treatment withanti-PD-L1 or MEK inhibitor treatment alone.

FIG. 7 demonstrates that MEK inhibitor treatment enhanced anti-tumoractivity of anti-PD-L1 antibodies in in vivo models of colorectalcancer. (A) Graph depicting changes in tumor volume with anti-PD-L1antibodies and MEK inhibitor co-treatment demonstrate a significantreduction of early stage tumor growth and sustained anti-tumor effect ascompared to anti-PD-L1 antibodies or MEK inhibitor treatment alone. (B)Graph depicting changes in tumor volume with anti-PD-L1 antibodies andMEK inhibitor co-treatment demonstrate a significant inhibition of latestage tumor growth as compared to anti-PD-L1 antibodies or MEK inhibitortreatment alone.

FIG. 8 is a series of graphs demonstrating that MEK inhibitor doses weremore effective when used in combination with anti-PD-L1 antibody fortreatment in in vivo models of colorectal cancer. (A) Graph depictingreduction in tumor volume with increasing doses of MEK inhibitorGDC-0973 treatment. (B) Graph depicting reduction in tumor volume uponadministration of anti-PD-L1 antibody in combination with differentdoses of MEK inhibitor GDC-0973. Mpk indicates milligrams per kilogram(mg/kg).

FIG. 9 is a graph demonstrating that treatment with MEK inhibitorG02443714 enhanced the anti-tumor activity of anti-PD-L1 antibodies inin vivo models of colorectal cancer. An enhanced reduction in tumorvolume with anti-PD-L1 antibody and MEK inhibitor combination treatmentwas observed as compared to treatment with anti-PD-L1 antibody or MEKinhibitor G02443714 alone.

FIG. 10 is a graph demonstrating that treatment with MEK inhibitorG02442104 enhanced the anti-tumor activity of anti-PD-L1 antibodies inin vivo models of colorectal cancer. An enhanced reduction in tumorvolume with anti-PD-L1 antibody and MEK inhibitor combination treatmentwas observed as compared to treatment with anti-PD-L1 antibody or MEKinhibitor G02442104 alone.

FIG. 11 is a graph demonstrating that treatment with MEK inhibitorG00039805 enhanced the anti-tumor activity of anti-PD-L1 antibodies inin vivo models of colorectal cancer. An enhanced reduction in tumorvolume with anti-PD-L1 antibody and MEK inhibitor combination treatmentwas observed as compared to treatment with anti-PD-L1 antibody or MEKinhibitor G00039805 alone.

FIG. 12 demonstrates that MEK inhibitor treatment enhanced anti-tumoractivity of anti-PD-L1 antibodies in in vivo models of melanoma. (A andB) Graph depicting changes in tumor volume with anti-PD-L1 antibodiesand MEK inhibitor co-treatment demonstrates significantly reduced tumorgrowth as compared to anti-PD-L1 antibodies or MEK inhibitor treatmentalone.

FIG. 13 is a graph demonstrating that co-treatment with anti-PD-L1antibodies and a chemotherapeutic agent Temodar did not reduce tumorgrowth in an in vivo model of melanoma. Therefore, the anti-tumor effectof MEK inhibitor and anti-PD-L1 antibodies is specific.

FIG. 14 is a graph demonstrating that co-treatment with anti-OX40antibodies and a MEK inhibitor did not reduce tumor growth in an in vivocolorectal model. Therefore, the anti-tumor effect of MEK inhibitor andanti-PD-L1 antibodies is specific.

FIG. 15 contains several graphs showing that MEK inhibitor increasedactivation of dendritic cells independently of anti-PD-L1 antibodytreatment. (A) Graph demonstrating that anti-PD-L1 antibody treatmentslightly increased MHC I surface expression. MEK inhibitor treatmentsignificantly enhanced MHCI expression, however co-treatment withanti-PD-L1 antibodies did not enhance the effect of MEK inhibitortreatment. (B-D) Graphs demonstrating that anti-PD-L1 antibody treatmentdid not increase expression of dendritic cell activation markers MHC II,CD80, and CD86. In contrast MEK inhibitor treatment significantlyenhanced expression of dendritic cell activation markers. Co-treatmentwith anti-PD-L1 antibodies did not enhance the effect of MEK inhibitortreatment. (E-H) Graphs demonstrating that stimulation of dendriticcells with anti-CD40 antibodies did not alter the effect of MEKinhibitor and anti-PD-L1 co-treatment on dendritic cell activation.

DETAILED DESCRIPTION OF THE INVENTION I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993).

II. Definitions

The term “PD-1 axis binding antagonist” is a molecule that inhibits theinteraction of a PD-1 axis binding partner with either one or more ofits binding partner, so as to remove T-cell dysfunction resulting fromsignaling on the PD-1 signaling axis with a result being to restore orenhance T-cell function (e.g., proliferation, cytokine production,target cell killing). As used herein, a PD-1 axis binding antagonistincludes a PD-1 binding antagonist, a PD-L1 binding antagonist and aPD-L2 binding antagonist.

The term “PD-1 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to itsbinding partners. In a specific aspect, the PD-1 binding antagonistinhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1binding antagonists include anti-PD-1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-1 withPD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reducesthe negative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes mediated signaling through PD-1 soas render a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, thePD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect,a PD-1 binding antagonist is MDX-1106 described herein. In anotherspecific aspect, a PD-1 binding antagonist is Merck 3745 describedherein. In another specific aspect, a PD-1 binding antagonist is CT-011described herein.

The term “PD-L1 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.S70 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. Instill another specific aspect, an anti-PD-L1 antibody is MPDL3280Adescribed herein.

The term “PD-L2 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to itsbinding partners. In a specific aspect, the PD-L2 binding antagonistinhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2antagonists include anti-PD-L2 antibodies, antigen binding fragmentsthereof, immunoadhesins, fusion proteins, oligopeptides and othermolecules that decrease, block, inhibit, abrogate or interfere withsignal transduction resulting from the interaction of PD-L2 with eitherone or more of its binding partners, such as PD-1. In one embodiment, aPD-L2 binding antagonist reduces the negative co-stimulatory signalmediated by or through cell surface proteins expressed on T lymphocytesmediated signaling through PD-L2 so as render a dysfunctional T-cellless dysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, a PD-L2 binding antagonist is animmunoadhesin.

The term “dysfunction” in the context of immune dysfunction, refers to astate of reduced immune responsiveness to antigenic stimulation. Theterm includes the common elements of both exhaustion and/or anergy inwhich antigen recognition may occur, but the ensuing immune response isineffective to control infection or tumor growth.

The term “dysfunctional”, as used herein, also includes refractory orunresponsive to antigen recognition, specifically, impaired capacity totranslate antigen recognition into down-stream T-cell effectorfunctions, such as proliferation, cytokine production (e.g., IL-2)and/or target cell killing.

The term “anergy” refers to the state of unresponsiveness to antigenstimulation resulting from incomplete or insufficient signals deliveredthrough the T-cell receptor (e.g. increase in intracellular Ca⁺² in theabsence of ras-activation). T cell anergy can also result uponstimulation with antigen in the absence of co-stimulation, resulting inthe cell becoming refractory to subsequent activation by the antigeneven in the context of costimulation. The unresponsive state can oftenbe overriden by the presence of Interleukin-2. Anergic T-cells do notundergo clonal expansion and/or acquire effector functions.

The term “exhaustion” refers to T cell exhaustion as a state of T celldysfunction that arises from sustained TCR signaling that occurs duringmany chronic infections and cancer. It is distinguished from anergy inthat it arises not through incomplete or deficient signaling, but fromsustained signaling. It is defined by poor effector function, sustainedexpression of inhibitory receptors and a transcriptional state distinctfrom that of functional effector or memory T cells. Exhaustion preventsoptimal control of infection and tumors. Exhaustion can result from bothextrinsic negative regulatory pathways (e.g., immunoregulatorycytokines) as well as cell intrinsic negative regulatory (costimulatory)pathways (PD-1, B7-H3, B7-H4, etc.).

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8⁺ T-cells,increased proliferation, increased antigen responsiveness (e.g., viral,pathogen, or tumor clearance) relative to such levels before theintervention. In one embodiment, the level of enhancement is as least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Themanner of measuring this enhancement is known to one of ordinary skillin the art.

A “T cell dysfunctional disorder” is a disorder or condition of T-cellscharacterized by decreased responsiVeness to antigenic stimulation. In aparticular embodiment, a T-cell dysfunctional disorder is a disorderthat is specifically associated with inappropriate increased signalingthrough PD-1. In another embodiment, a T-cell dysfunctional disorder isone in which T-cells are anergic or have decreased ability to secretecytokines, proliferate, or execute cytolytic activity. In a specificaspect, the decreased responsiveness results in ineffective control of apathogen or tumor expressing an immunogen. Examples of T celldysfunctional disorders characterized by T-cell dysfunction includeunresolved acute infection, chronic infection and tumor immunity.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.

“Immunogenecity” refers to the ability of a particular substance toprovoke an immune response. Tumors are immunogenic and enhancing tumorimmunogenicity aids in the clearance of the tumor cells by the immuneresponse. Examples of enhancing tumor immunogenicity include treatmentwith anti-PDL antibodies and a MEK inhibitor.

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration at least the same as the treatmentduration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatmentduration.

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies(e.g., bispecific antibodies, diabodies, and single-chain molecules, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv). The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for 1.1 and E isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains. Depending on the amino acid sequence of the constantdomain of their heavy chains (CH), immunoglobulins can be assigned todifferent classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chainsdesignated α, δ, ε, γ and pt, respectively. The γ and a classes arefurther divided into subclasses on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “VH” and “VL”, respectively. These domains are generally the mostvariable parts of the antibody (relative to other antibodies of the sameclass) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments calledhypervariable regions (HVRs) both in the light-chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast topolyclonal antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler andMilstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2^(nd) ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhuet al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851(1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The term “naked antibody” refers to an antibody that is not conjugatedto a cytotoxic moiety or radiolabel.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antibody fragment. Specifically wholeantibodies include those with heavy and light chains including an Fcregion. The constant domains may be native sequence constant domains(e.g., human native sequence constant domains) or amino acid sequencevariants thereof. In some cases, the intact antibody may have one ormore effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments. Papain digestion of antibodies produced twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (V_(H)), and thefirst constant domain of one heavy chain (C_(H)1). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having different antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having a few additional residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“FV” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of the antibodies of the invention comprise aportion of an intact antibody, generally including the antigen bindingor variable region of the intact antibody or the Fc region of anantibody which retains or has modified FcR binding capability. Examplesof antibody fragments include linear antibody, single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). Chimeric antibodies of interest herein include PRIMATIZED®antibodies wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by, e.g., immunizing macaque monkeys with anantigen of interest. As used herein, “humanized antibody” is used asubset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR(hereinafter defined) of the recipient are replaced by residues from anHVR of a non-human species (donor antibody) such as mouse, rat, rabbitor non-human primate having the desired specificity, affinity, and/orcapacity. In some instances, framework (“FR”) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, etc. The number of these aminoacid substitutions in the FR are typically no more than 6 in the Hchain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is an antibody that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in i Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available compleX crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

A “human consensus framework” or “acceptor human framework” is aframework that represents the most commonly occurring amino acidresidues in a selection of human immunoglobulin VL or VH frameworksequences. Generally, the selection of human immunoglobulin VL or VHsequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al., Sequences ofProteins of Immunological Interest, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Examples includefor the VL, the subgroup may be subgroup kappa I, kappa II, kappa III orkappa IV as in Kabat et al., supra. Additionally, for the VH, thesubgroup may be subgroup I, subgroup II, or subgroup III as in Kabat etal., supra. Alternatively, a human consensus framework can be derivedfrom the above in which particular residues, such as when a humanframework residue is selected based on its homology to the donorframework by aligning the donor framework sequence with a collection ofvarious human framework sequences. An acceptor human framework “derivedfrom” a human immunoglobulin framework or a human consensus frameworkmay comprise the same amino acid sequence thereof, or it may containpre-existing amino acid sequence changes. In some embodiments, thenumber of pre-existing amino acid changes are 10 or less, 9 or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1) (SEQID NO:4), WVRQAPGKGLEWV (HC-FR2), (SEQ ID NO:5),RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (HC-FR3, SEQ ID NO:6), WGQGTLVTVSA(HC-FR4), (SEQ ID NO:7).

A “VL kappa I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: DIQMTQSPSSLSASVGDRVTITC (LC-FR1) (SEQID NO:11), WYQQKPGKAPKLLIY (LC-FR2) (SEQ ID NO:12),GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (LC-FR3) (SEQ ID NO:13), FGQGTKVEIKR(LC-FR4) (SEQ ID NO:14).

An “amino-acid modification” at a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent thespecified residue. Insertion “adjacent” to a specified residue meansinsertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue. The preferred aminoacid modification herein is a substitution.

An “affinity-matured” antibody is one with one or more alterations inone or more HVRs thereof that result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by VH— and VL-domain shuffling. Randommutagenesis of HVR and/or framework residues is described by, forexample: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As use herein, the term “specifically binds to” or is “specific for”refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds to a target (which can be an epitope) is an antibodythat binds this target with greater affinity, avidity, more readily,and/or with greater duration than it binds to other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that specifically binds to a target has a dissociation constant(Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certainembodiments, an antibody specifically binds to an epitope on a proteinthat is conserved among the protein from different species. In anotherembodiment, specific binding can include, but does not require exclusivebinding.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2 (including IgG2A and IgG2B), IgG-3, or IgG-4 subtypes, IgA(including IgA-1 and IgA-2), IgE, IgD or IgM. The Ig fusions preferablyinclude the substitution of a domain of a polypeptide or antibodydescribed herein in the place of at least one variable region within anIg molecule. In a particularly preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3regions of an IgG1 molecule. For the production of immunoglobulinfusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995. Forexample, useful immunoadhesins as second medicaments useful forcombination therapy herein include polypeptides that comprise theextracellular or PD-1 binding portions of PD-L1 or PD-L2 or theextracellular or PD-L1 or PD-L2 binding portions of PD-1, fused to aconstant domain of an immunoglobulin sequence, such as a PD-L1 ECD Fc, aPD-L2 ECD Fc, and a PD-1 ECD-Fc, respectively.

Immunoadhesin combinations of Ig Fc and ECD of cell surface receptorsare sometimes termed soluble receptors.

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving two portions covalently linked together, where each of theportions is a polypeptide having a different property. The property maybe a biological property, such as activity in vitro or in vivo. Theproperty may also be simple chemical or physical property, such asbinding to a target molecule, catalysis of a reaction, etc. The twoportions may be linked directly by a single peptide bond or through apeptide linker but are in reading frame with each other.

A “PD-1 oligopeptide,” “PD-L1 oligopeptide,” or “PD-L2 oligopeptide” isan oligopeptide that binds, preferably specifically, to a PD-1, PD-L1 orPD-L2 negative costimulatory polypeptide, respectively, including areceptor, ligand or signaling component, respectively, as describedherein. Such oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. Such oligopeptides are usually at least about 5amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100amino acids in length or more. Such oligopeptides may be identifiedusing well known techniques. In this regard, it is noted that techniquesfor screening oligopeptide libraries for oligopeptides that are capableof specifically binding to a polypeptide target are well known in theart (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT PublicationNos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci.USA., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A.,82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens,130-149 (1986); Geysen et al., I Immunol. Meth., 102:259-274 (1987);Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al.Proc. Natl. Acad. Sci. USA, 87:6378 (1990); Lowman, H. B. et al.Biochemistry, 30:10832 (1991); Clackson, T. et al. Nature, 352: 624(1991); Marks, J. D. et al., J. Mol. Biol., 222:581 (1991); Kang, A. S.et al. Proc. Natl. Acad. Sci. USA, 88:8363 (1991), and Smith, G. P.,Current Opin. Biotechnol., 2:668 (1991).

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen. Theanti-PD-L1 antibodies of the invention block the signaling through PD-1so as to restore a functional response by T-cells (e.g., proliferation,cytokine production, target cell killing) from a dysfunctional state toantigen stimulation.

An “agonist” or activating antibody is one that enhances or initiatessignaling by the antigen to which it binds. In some embodiments, agonistantibodies cause or activate signaling without the presence of thenatural ligand.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG 1, IgG2 (IgG2A,IgG2B), IgG3 and IgG4.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see M. Daëron, Annu.Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J.Immunol. 24: 249 (1994). Methods of measuring binding to FcRn are known(see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997);Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton etal., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004/92219 (Hinton etal.). Binding to FcRn in vivo and serum half-life of human FcRnhigh-affinity binding polypeptides can be assayed, e.g., in transgenicmice or transfected human cell lines expressing human FcRn, or inprimates to which the polypeptides having a variant Fc region areadministered. WO 2004/42072 (Presta) describes antibody variants whichimproved or diminished binding to FcRs. See also, e.g., Shields et al.,J. Biol. Chem. 9(2): 6591-6604 (2001).

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™

A “package insert” refers to instructions customarily included incommercial packages of medicaments that contain information about theindications customarily included in commercial packages of medicamentsthat contain information about the indications, usage, dosage,administration, contraindications, other medicaments to be combined withthe packaged product, and/or warnings concerning the use of suchmedicaments, etc.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” if oneor more symptoms associated with cancer are mitigated or eliminated,including, but are not limited to, reducing the proliferation of (ordestroying) cancerous cells, decreasing symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, delaying the progression of the disease, and/or prolongingsurvival of individuals.

As used herein, “delaying progression of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

An “effective amount” is at least the minimum concentration required toeffect a measurable improvement or prevention of a particular disorder.An effective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe antibody to elicit a desired response in the individual. Aneffective amount is also one in which any toxic or detrimental effectsof the treatment are outweighed by the therapeutically beneficialeffects. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as decreasing one or more symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, enhancing effect of another medicationsuch as via targeting, delaying the progression of the disease, and/orprolonging survival. In the case of cancer or tumor, an effective amountof the drug may have the effect in reducing the number of cancer cells;reducing the tumor size; inhibiting (i.e., slow to some extent ordesirably stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and desirably stop) tumor metastasis;inhibiting to some extent tumor growth; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

As used herein, “complete response” or “CR” refers to disappearance ofall target lesions; “partial response” or “PR” refers to at least a 30%decrease in the sum of the longest diameters (SLD) of target lesions,taking as reference the baseline SLD; and “stable disease” or “SD”refers to neither sufficient shrinkage of target lesions to qualify forPR, nor sufficient increase to qualify for PD, taking as reference thesmallest SLD since the treatment started.

As used herein, “progressive disease” or “PD” refers to at least a 20%increase in the SLD of target lesions, taking as reference the smallestSLD recorded since the treatment started or the presence of one or morenew lesions.

As used herein, “progression free survival” (PFS) refers to the lengthof time during and after treatment during which the disease beingtreated (e.g., cancer) does not get worse. Progression-free survival mayinclude the amount of time patients have experienced a complete responseor a partial response, as well as the amount of time patients haveexperienced stable disease.

As used herein, “overall response rate” (ORR) refers to the sum ofcomplete response (CR) rate and partial response (PR) rate.

As used herein, “overall survival” refers to the percentage ofindividuals in a group who are likely to be alive after a particularduration of time.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan, and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed;callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesinsynthetic analogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4integrin inhibitor; a sarcodictyin; spongistatin; nitrogen mustards suchas chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such asthe enediyne antibiotics (e.g., calicheamicin, especially calicheamicingammalI and calicheamicin omega11 (see, e.g., Nicolaou et al., Angew.Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®) and deoxydoxorubicin), epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine(XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acidanalogues such as denopterin, methotrexate, pteropterin, trimetrexate;purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine,thioguanine; pyrimidine analogs such as ancitabine, azacitidine,6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidinederivative), as well as other c-Kit inhibitors; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK®polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®,FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g.,paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation ofpaclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin;leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Additional examples of chemotherapeutic agents include anti-hormonalagents that act to regulate, reduce, block, or inhibit the effects ofhormones that can promote the growth of cancer, and are often in theform of systemic, or whole-body treatment. They may be hormonesthemselves. Examples include anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (FARESTON®); anti-progesterones; estrogen receptordown-regulators (ERDs); estrogen receptor antagonists such asfulvestrant (FASLODEX®); agents that function to suppress or shut downthe ovaries, for example, leutinizing hormone-releasing hormone (LHRH)agonists such as leuprolide acetate (LUPRON® and ELIGARD®), goserelinacetate, buserelin acetate and tripterelin; anti-androgens such asflutamide, nilutamide and bicalutamide; and aromatase inhibitors thatinhibit the enzyme aromatase, which regulates estrogen production in theadrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMASIN®),formestanie, fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), andanastrozole (ARIMIDEX®). In addition, such definition ofchemotherapeutic agents includes bisphosphonates such as clodronate (forexample, BONEFOS® or OSTAC®), etidronate (DIDROCAL8), NE-58095,zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®),pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);anti-sense oligonucleotides, particularly those that inhibit expressionof genes in signaling pathways implicated in abherant cellproliferation, such as, for example, PKC-alpha, Raf, H-Ras, andepidermal growth factor receptor (EGF-R); vaccines such as THERATOPE®vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine,LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g.,LURTOTECAN®); an anti-estrogen such as fulvestrant; a Kit inhibitor suchas imatinib or EXEL-0862 (a tyrosine kinase inhibitor); EGFR inhibitorsuch as erlotinib or cetuximab; an anti-VEGF inhibitor such asbevacizumab; arinotecan; rmRH (e.g., ABARELIX®); lapatinib and lapatinibditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-moleculeinhibitor also known as GW572016); 17AAG (geldanamycin derivative thatis a heat shock protein (Hsp) 90 poison), and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

As used herein, the term “cytokine” refers generically to proteinsreleased by one cell population that act on another cell asintercellular mediators or have an autocrine effect on the cellsproducing the proteins. Examples of such cytokines include lymphokines,monokines; interleukins (“ILs”) such as IL-1, IL-1α, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-15,IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN®rIL-2; a tumor-necrosis factor such as TNF-α or TNF-β, TGF-β1-3; andother polypeptide factors including leukemia inhibitory factor (“LIF”),ciliary neurotrophic factor (“CNTF”), CNTF-like cytokine (“CLC”),cardiotrophin (“CT”), and kit ligand (“KL”).

As used herein, the term “chemokine” refers to soluble factors (e.g.,cytokines) that have the ability to selectively induce chemotaxis andactivation of leukocytes. They also trigger processes of angiogenesis,inflammation, wound healing, and tumorigenesis. Example chemokinesinclude IL-8, a human homolog of murine keratinocyte chemoattractant(KC).

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms. Examples of alkyl groups include, but are not limited to, methyl(Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃),2-propyl (i-Pr, i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl,—CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl(s-Bu, s-butyl, —CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl,—C(CH₃)₃), 1-pentyl (n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl(—CH(CH₃)CH₂CH₂CH₃), 3-pentyl (—CH(CH₂CH₃)₂), 2-methyl-2-butyl(—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl (—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl(—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl (—CH₂CH(CH₃)CH₂CH₃), 1-hexyl(—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl (—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl(—CH(CH₂CH₃)(CH₂CH₂CH₃)), 2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃),3-methyl-2-pentyl (—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl(—CH(CH₃)CH₂CH(CH₃)₂), 3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂),2-methyl-3-pentyl (—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl(—C(CH₃)₂CH(CH₃)₂), 3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl,1-octyl, and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to twelve carbon atoms with at least one siteof unsaturation, i.e., a carbon-carbon, sp² double bond, wherein thealkenyl radical includes radicals having “cis” and “trans” orientations,or alternatively, “E” and “Z” orientations. Examples include, but arenot limited to, ethylenyl or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), andthe like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to twelve carbon atoms with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond. Examples include,but are not limited to, ethynyl propynyl (propargyl, —CH₂C≡CH), and thelike.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms as a monocyclic ring or 7to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to12 atoms can be arranged, for example, as a bicyclo [4,5], [5,5], [5,6]or [6,6] system, and bicyclic carbocycles having 9 or 10 ring atoms canbe arranged as a bicyclo [5,6] or [6,6] system, or as bridged systemssuch as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-18 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Some aryl groups are representedin the exemplary structures as “Ar”. Aryl includes bicyclic radicalscomprising an aromatic ring fused to a saturated, partially unsaturatedring, or aromatic carbocyclic or heterocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, indenyl, indanyl,1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 18 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen and sulfur,the remaining ring atoms being C, where one or more ring atoms isoptionally substituted independently with one or more substituentsdescribed below. A heterocycle may be a monocycle having 3 to 7 ringmembers (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O,P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atomsand 1 to 6 heteroatoms selected from N, O, P, and S), for example: abicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocycles are describedin Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and9; “The Chemistry of Heterocyclic Compounds, A series of Monographs”(John Wiley & Sons, New York, 1950 to present), in particular Volumes13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.“Heterocyclyl” also includes radicals where heterocycle radicals arefused with a saturated, partially unsaturated ring, or aromaticcarbocyclic or heterocyclic ring. Examples of heterocyclic ringsinclude, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, and azabicyclo[2.2.2]hexanyl. Spiromoieties are also included within the scope of this definition. Examplesof a heterocyclic group wherein ring atoms are substituted with oxo (═O)moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl.

The term “heteroaryl” refers to a monovalent aromatic radical of 5- or6-membered rings, and includes fused ring systems (at least one of whichis aromatic) of 5-18 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl.

The heterocycle or heteroaryl groups may be carbon (carbon-linked) ornitrogen (nitrogen-linked) attached where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

The heteroatoms present in heteroaryl or heterocyclcyl include theoxidized forms such as N⁺→O⁻, S(O) and S(O)₂.

The term “halo” refers to F, Cl, Br or I.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid; such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid; and ethanolamine.The term “hydrate” refers to the complex where the solvent molecule iswater.

It is understood that aspects and variations of the invention describedherein include “consisting of” and/or “consisting essentially of”aspects and variations.

III Methods

In one aspect, provided herein is a method for treating or delayingprogression of cancer in an individual comprising administering to theindividual an effective amount of a PD-1 axis binding antagonist and aMEK inhibitor. In some embodiments, the treatment results in sustainedresponse in the individual after cessation of the treatment.

The methods of this invention may find use in treating conditions whereenhanced immunogenicity is desired such as increasing tumorimmunogenicity for the treatment of cancer. A variety of cancers may betreated, or their progression may be delayed, including but are notlimited to a cancer that may contain a BRAF V600E mutation, a cancerthat may contain a BRAF wildtype, a cancer that may contain a KRASwildtype, or a cancer that may contain an activating KRAS mutation.

In some embodiments, the individual has melanoma. The melanoma may be atearly stage or at late stage. In some embodiments, the individual hascolorectal cancer. The colorectal cancer may be at early stage or atlate stage. In some embodiments, the individual has non-small cell lungcancer. The non-small cell lung cancer may be at early stage or at latestage. In some emodiements, the individual has pancreatic cancer. Thepancreatice cancer may be at early stage or late state. In someembodiments, the individual has a hematological malignancy. Thehematological malignancy may be early stage or late stage. In someembodiments, the individual has ovarian cancer. The ovarian cancer maybe at early stage or at late stage. In some embodiments, the individualhas breast cancer. The breast cancer may be at early stage or at latestage. In some embodiments, the individual has renal cell carcinoma. Therenal cell carcinoma may be at early stage or at late stage.

In some embodiments, the individual is a mammal, such as domesticatedanimals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g.,humans and non-human primates such as monkeys), rabbits, and rodents(e.g., mice and rats). In some embodiments, the individual treated is ahuman.

In another aspect, provided herein is a method of enhancing immunefunction in an individual having cancer comprising administering aneffective amount of a PD-1 axis binding antagonist and a MEK inhibitor.

In some embodiments, the CD8 T cells in the individual have enhancedpriming, activation, proliferation and/or cytolytic activity relative toprior to the administration of the PD-1 pathway antagonist and the MEKinhibitor. In some embodiments, the CD8 T cell priming is characterizedby elevated CD44 expression and/or enhanced cytolytic activity in CD8 Tcells. In some embodiments, the CD8 T cell activation is characterizedby an elevated frequency of γ-IFN⁺ CD8 T cells. In some embodiments, theCD8 T cell is an antigen-specific T-cell. In some embodiments, theimmune evasion by signaling through PD-L1 surface expression isinhibited.

In some embodiments, the cancer cells in the individual have elevatedexpression of MHC class I antigen expression relative to prior to theadministration of the PD-1 pathway antagonist and the MEK inhibitor.

In some embodiment's, the antigen presenting cells in the individualhave enhanced maturation and activation relative prior to theadministration of the PD-1 pathway antagonist and the MEK inhibitor. Insome embodiments, wherein the antigen presenting cells are dendriticcells. In some embodiments, the maturation of the antigen presentingcells is characterized by increased frequency of CD83⁺ dendritic cells.In some embodiments, the activation of the antigen presenting cells ischaracterized by elevated expression of CD80 and CD86 on dendriticcells.

In some embodiments, the serum levels of cytokine IL-10 and/or chemokineIL-8, a human homolog of murine KC, in the individual are reducedrelative prior to the administration of the anti-PD-L1 antibody and theMEK inhibitor.

In some embodiments, the cancer has elevated levels of T-cellinfiltration.

In some embodiments, the combination therapy of the invention comprisesadministration of a PD-1 axis binding antagonist and a MEK inhibitor.The PD-1 axis binding antagonist and the MEK inhibitor may beadministered in any suitable manner known in the art. For example, ThePD-1 axis binding antagonist and the MEK inhibitor may be administeredsequentially (at different times) or concurrently (at the same time).

In some embodiments, the MEK inhibitor is administered continuously. Insome embodiments, the MEK inhibitor is administered intermittently. Insome embodiments, the MEK inhibitor is administered beforeadministration of the PD-1 axis binding antagonist. In some embodiments,the MEK inhibitor is administered simultaneously with administration ofthe PD-1 axis binding antagonist. In some embodiments, the MEK inhibitoris administered after administration of the PD-1 axis bindingantagonist.

In some embodiments, provided is a method for treating or delayingprogression of cancer in an individual comprising administering to theindividual an effective amount of a PD-1 axis binding antagonist and aMEK inhibitor, further comprising administering an additional therapy.The additional therapy may be radiation therapy, surgery (e.g.,lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy,viral therapy, RNA therapy, immunotherapy, bone marrow transplantation,nanotherapy, monoclonal antibody therapy, or a combination of theforegoing. The additional therapy may be in the form of adjuvant orneoadjuvant therapy. In some embodiments, the additional therapy is theadministration of small molecule enzymatic inhibitor or anti-metastaticagent. In some embodiments, the additional therapy is the administrationof side-effect limiting agents (e.g., agents intended to lessen theoccurrence and/or severity of side effects of treatment, such asanti-nausea agents, etc.). In some embodiments, the additional therapyis radiation therapy. In some embodiments, the additional therapy issurgery. In some embodiments, the additional therapy is a combination ofradiation therapy and surgery. In some embodiments, the additionaltherapy is gamma irradiation. In some embodiments, the additionaltherapy is therapy targeting PI3K/AKT/mTOR pathway, HSP90 inhibitor,tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent.The additional therapy may be one or more of the chemotherapeutic agentsdescribed hereabove.

The PD-1 axis binding antagonist and the MEK inhibitor may beadministered by the same route of administration or by different routesof administration. In some embodiments, the PD-1 axis binding antagonistis administered intravenously, intramuscularly, subcutaneously,topically, orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally. In some embodiments, the MEK inhibitor is administeredintravenously, intramuscularly, subcutaneously, topically, orally,transdermally, intraperitoneally, intraorbitally, by implantation, byinhalation, intrathecally, intraventricularly, or intranasally. Aneffective amount of the PD-1 axis binding antagonist and the MEKinhibitor may be administered for prevention or treatment of disease.The appropriate dosage of the PD-1 axis binding antagonist and/or theMEK inhibitor may be deterimined based on the type of disease to betreated, the type of the PD-1 axis binding antagonist and the MEKinhibitor, the severity and course of the disease, the clinicalcondition of the individual, the individual's clinical history andresponse to the treatment, and the discretion of the attendingphysician.

Any of the PD-1 axis binding antagonists and the MEK inhibitors known inthe art or described below may be used in the methods.

PD-1 Axis Binding Antagonists

Provided herein is a method for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and a MEK inhibitor.For example, a PD-1 axis binding antagonist includes a PD-1 bindingantagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.Alternative names for “PD-1” include CD279 and SLEB2. Alternative namesfor “PD-L1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for“PD-L2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1,PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect the PD-1 ligand binding partners are PD-L1 and/or PD-L2.In another embodiment, a PD-L1 binding antagonist is a molecule thatinhibits the binding of PD-L1 to its binding partners. In a specificaspect, PD-L1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PD-L2 binding antagonist is a molecule that inhibits thebinding of PD-L2 to its binding partners. In a specific aspect, a PD-L2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide.

In some embodiment, the PD-1 binding antagonist is an anti-PD-1 antibody(e.g., a human antibody, a humanized antibody, or a chimeric antibody).In some embodiments, the anti-PD-1 antibody is selected from the groupconsisting of MDX-1106, Merck 3475 and CT-011. In some embodiments, thePD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesincomprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2fused to a constant region (e.g., an Fc region of an immunoglobulinsequence). In some embodiments, the PD-1 binding antagonist is AMP-224.In some embodiments, the PD-L1 binding antagonist is anti-PD-L1antibody. In some embodiments, the anti-PD-L1 binding antagonist isselected from the group consisting of YW243.55.S70, MPDL3280A andMDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibodydescribed in WO2007/005874. Antibody YW243.55.S70 (heavy and light chainvariable region sequences shown in SEQ ID Nos. 20 and 21, respectively)is an anti-PD-L1 described in WO 2010/077634 A1. MDX-1106, also known asMDX-1106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody describedin WO2006/121168. Merck 3745, also known as MK-3475 or SCH-900475, is ananti-PD-1 antibody described in WO2009/114335. CT-011, also known ashBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611.AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptordescribed in WO2010/027827 and WO2011/066342.

In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternativenames for “MDX-1106” include MDX-1106-04, ONO-4538, BMS-936558 orNivolumab. In some embodiments, the anti-PD-1 antibody is Nivolumab (CASRegistry Number: 946414-94-4). In a still further embodiment, providedis an isolated anti-PD-1 antibody comprising a heavy chain variableregion comprising the heavy chain variable region amino acid sequencefrom SEQ ID NO:22 and/or a light chain variable region comprising thelight chain variable region amino acid sequence from SEQ ID NO:23. In astill further embodiment, provided is an isolated anti-PD-1 antibodycomprising a heavy chain and/or a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:

(SEQ ID NO: 22) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEELGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK, or

(b) the light chain sequences has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 23) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Examples of anti-PD-L1 antibodies useful for the methods of thisinvention, and methods for making thereof are described in PCT patentapplication WO 2010/077634 A1, which is incorporated herein byreference.

In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1antibody. In some embodiments, the anti-PD-L1 antibody is capable ofinhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1.In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody.In some embodiments, the anti-PD-L1 antibody is an antibody fragmentselected from the group consisting of Fab, Fab′-SH, Fv, scFv, and(Fab′)₂ fragments. In some embodiments, the anti-PD-L1 antibody is ahumanized antibody. In some embodiments, the anti-PD-L1 antibody is ahuman antibody.

The anti-PD-L1 antibodies useful in this invention, includingcompositions containing such antibodies, such as those described in WO2010/077634 A1, may be used in combination with a MEK inhibitor to treatcancer. In some embodiments, the anti-PD-L1 antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:20and a light chain variable region comprising the amino acid sequence ofSEQ ID NO:21.

In one embodiment, the anti-PD-L1 antibody contains a heavy chainvariable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3sequence, wherein:

(a) the HVR-H1 sequence is GFTFSX₁SWIH (SEQ ID NO:1);

(b) the HVR-H2 sequence is AWIX₂PYGGSX₃YYADSVKG (SEQ ID NO:2);

(c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:3);

further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S.

In one specific aspect, X₁ is D; X₂ is S and X₃ is T. In another aspect,the polypeptide further comprises variable region heavy chain frameworksequences juxtaposed between the HVRs according to the formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the framework sequences are VHsubgroup III consensus framework. In a still further aspect, at leastone of the framework sequences is the following:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:4)

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:5)

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:6)

HC-FR4 is WGQGTLVTVSA (SEQ ID NO:7).

In a still further aspect, the heavy chain polypeptide is furthercombined with a variable region light chain comprising an HVR-L1, HVR-L2and HVR-L3, wherein:

(a) the HVR-L1 sequence is RASQX₄X₅X₆TX₇X₈A (SEQ ID NO:8);

(b) the HVR-L2 sequence is SASX₉LX₁₀S, (SEQ ID NO:9);

(c) the HVR-L3 sequence is QQX₁₁X₁₂X₁₃X₁₄PX₁₅T (SEQ ID NO:10);

-   -   further wherein: X₄ is D or V; X₅ is V or I; X₆ is S or N; X₇ is        A or F; X₈ is V or L; X₉ is F or T; X₁₀ is Y or A; X₁ is Y, G,        F, or S; X₁₂ is L, Y, F or W; X₁₃ is Y, N, A, T, G, F or I; X₁₄        is H, V, P, T or I; X₁₅ is A, W, R, P or T.

In a still further aspect, X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V;X₉ is F; X₁₀ is Y; X₁₁ is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H; X₁₅ is A. Ina still further aspect, the light chain further comprises variableregion light chain framework sequences juxtaposed between the HVRsaccording to the formula:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the frameworksequences are VL kappa I consensus framework. In a still further aspect,at least one of the framework sequence is the following:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:11)

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO:12)

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:13)

LC-FR4 is FGQGTKVEIKR (SEQ ID NO:14).

In another embodiment, provided is an isolated anti-PD-L1 antibody orantigen binding fragment comprising a heavy chain and a light chainvariable region sequence, wherein:

(a) the heavy chain comprises and HYR-1-H1, HVR-1-H2 and HVR-H3, whereinfurther:

-   -   (i) the HVR-H1 sequence is GFTFSX_(I)SWIH; (SEQ ID NO:1)    -   (ii) the HVR-H2 sequence is AWIX₂PYGGSX₃YYADSVKG (SEQ ID NO:2)    -   (iii) the HVR-H3 sequence is RHWPGGFDY, and (SEQ ID NO:3)

(b) the light chain comprises and HVR-L1, HVR-L2 and HVR-L3, whereinfurther:

-   -   (i) the HVR-L1 sequence is RASQX₄X₅X₆TX₇X₈A (SEQ ID NO:8)    -   (ii) the HVR-L2 sequence is SASX₉LX₁₀S; and (SEQ ID NO:9)    -   (iii) the HVR-L3 sequence is QQX₁₁X₁₂X₁₃X₁₄PX₁₅T; (SEQ ID NO:10)    -   Further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S; X₄ is        D or V; X₅ is V or 1; X₆ is S or N; X₇ is A or F; X₈ is V or L;        X₉ is F or T; X₁₀ is Y or A; X₁₁ is Y, G, F, or S; X₁₂ is L, Y,        F or W; X₁₃ is Y, N, A, T, G, F or 1; X₁₄ is H, V, P, T or I;        X₁₅ is A, W, R, P or T.

In a specific aspect, X₁ is D; X₂ is S and X₃ is T. In another aspect,X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V; X₉ is F; X₁₀ is Y; X_(I),is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H; X₁₅ is A. In yet another aspect, X₁is D; X₂ is S and X₃ is T, X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V;X₉ is F; X₁₀ is Y; X₁₁ is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H and X₁₅ is A.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences is thefollowing:

(SEQ ID NO: 4) HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 5)HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR  (SEQ ID NO: 7)HC-FR4 WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

(SEQ ID NO: 11) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 12)LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC  (SEQ ID NO: 14)LC-FR4 FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG 1. In a still further aspect, the murine constant regionis selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PD-L1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

-   -   (a) the heavy chain further comprises and HVR-H1, HVR-H2 and an        HVR-H3 sequence having at least 85% sequence identity to        GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16) and        RHWPGGFDY (SEQ ID NO:3), respectively, or    -   (b) the light chain further comprises an HVR-L1, HVR-L2 and an        HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18) and QQYLYHPAT        (SEQ ID NO:19), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(LC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a still further aspect, the heavy chainframework sequences are derived from a Kabat subgroup 1, II, or IIIsequence. In a still further aspect, the heavy chain framework sequenceis a VH subgroup III consensus framework. In a still further aspect, oneor more of the heavy chain framework sequences is the following:

(SEQ ID NO: 4) HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 5)HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR  (SEQ ID NO: 7)HC-FR4 WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

(SEQ ID NO: 11) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 12)LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC  (SEQ ID NO: 14)LC-FR4 FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In a still further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWG QGTLVTVSA (SEQID NO:20), or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ IDNO:21).

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences is the following:

(SEQ ID NO: 4) HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 5)HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR  (SEQ ID NO: 7)HC-FR4 WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

(SEQ ID NO: 11) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 12)LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC  (SEQ ID NO: 14)LC-FR4 FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG 1. In a still further aspect, the murine constant regionis selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In another further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chainsequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWG QGTLVTVSS (SEQID NO:24), or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ IllNO:21).

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences is the following:

(SEQ ID NO: 4) HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 5)HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR  (SEQ ID NO: 25)HC-FR4 WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

(SEQ ID NO: 11) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 12)LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC  (SEQ ID NO: 14)LC-FR4 FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In yet another embodiment, the anti-PD-1 antibody is MPDL3280A. In astill further embodiment, provided is an isolated anti-PD-1 antibodycomprising a heavy chain variable region comprising the heavy chainvariable region amino acid sequence from SEQ ID NO:24 and/or a lightchain variable region comprising the light chain variable region aminoacid sequence from SEQ ID NO:25. In a still further embodiment, providedis an isolated anti-PD-1 antibody comprising a heavy chain and/or alight chain sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:

(SEQ ID NO: 26)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, or

(b) the light chain sequences has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 27)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In a still further embodiment, the invention provides for compositionscomprising any of the above described anti-PD-L1 antibodies incombination with at least one pharmaceutically-acceptable carrier.

In a still further embodiment, provided is an isolated nucleic acidencoding a light chain or a heavy chain variable region sequence of ananti-PD-L1 antibody, wherein:

-   -   (a) the heavy chain further comprises and HVR-H1, HVR-H2 and an        HVR-H3 sequence having at least 85% sequence identity to        GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16) and        RHWPGGFDY (SEQ ID NO:3), respectively, and    -   (b) the light chain further comprises an HVR-L1, HVR-L2 and an        HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18) and QQYLYHPAT        (SEQ ID NO:19), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In aspect, theheavy chain variable region comprises one or more framework sequencesjuxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as: (LC-FR1)-(HVR-LI)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect,the framework sequences are derived from human consensus frameworksequences. In a further aspect, the heavy chain framework sequences arederived from a Kabat subgroup I, II, or III sequence. In a still furtheraspect, the heavy chain framework sequence is a VH subgroup IIIconsensus framework. In a still further aspect, one or more of the heavychain framework sequences is the following:

(SEQ ID NO: 4) HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 5)HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR  (SEQ ID NO: 7)HC-FR4 WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

(SEQ ID NO: 11) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 12)LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC  (SEQ ID NO: 14)LC-FR4 FGQGTKVEIKR.

In a still further specific aspect, the antibody described herein (suchas an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2antibody) further comprises a human or murine constant region. In astill further aspect, the human constant region is selected from thegroup consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still furtherspecific aspect, the human constant region is IgG1. In a still furtheraspect, the murine constant region is selected from the group consistingof IgG1, IgG2A, IgG2B, IgG3. In a still further aspect, the murineconstant region if IgG2A. In a still further specific aspect, theantibody has reduced or minimal effector function. In a still furtherspecific aspect, the minimal effector function results from productionin prokaryotic cells. In a still further specific aspect the minimaleffector function results from an “effector-less Fc mutation” oraglycosylation. In still a further aspect, the effector-less Fc mutationis an N297A or D265A/N297A substitution in the constant region.

In a still further aspect, provided herein are nucleic acids encodingany of the antibodies described herein. In some embodiments, the nucleicacid further comprises a vector suitable for expression of the nucleicacid encoding any of the previously described anti-PD-L1, anti-PD-1, oranti-PD-L2 antibodies. In a still further specific aspect, the vectorfurther comprises a host cell suitable for expression of the nucleicacid. In a still further specific aspect, the host cell is a eukaryoticcell or a prokaryotic cell. In a still further specific aspect, theeukaryotic cell is a mammalian cell, such as Chinese Hamster Ovary(CHO).

The antibody or antigen binding fragment thereof, may be made usingmethods known in the art, for example, by a process comprising culturinga host cell containing nucleic acid encoding any of the previouslydescribed anti-PD-L1, anti-PD-1, or anti-PD-L2 antibodies orantigen-binding fragment in a form suitable for expression, underconditions suitable to produce such antibody or fragment, and recoveringthe antibody or fragment.

In a still further embodiment, the invention provides for a compositioncomprising an anti-PD-L1, an anti-PD-1, or an anti-PD-L2 antibody orantigen binding fragment thereof as provided herein and at least onepharmaceutically acceptable carrier. In some embodiments, theanti-PD-L1, anti-PD-1, or anti-PD-L2 antibody or antigen bindingfragment thereof administered to the individual is a compositioncomprising one or more pharmaceutically acceptable carrier. Any of thepharmaceutically acceptable carrier described herein or known in the artmay be used.

MEK Inhibitors

The invention provides methods for treating cancer or slowingprogression of cancer in an individual comprising administering aneffective amount of a PD-1 pathway antagonist and a MEK inhibitor. Anyknown MEK inhibitors are intended, such as the MEK inhibitor compoundsdescribed in PCT patent applications WO 03/077914 A1, WO 2005/121142 A1,WO 2007/044515 A1, WO 2008/024725 A1 and WO 2009/085983 A 1, the contentof which are incorporated herein by reference. The MEK inhibitoradministered may be in a pharmaceutical composition or formulation. Insome embodiments, the pharmaceutical composition or formulationcomprises one or more MEK inhibitors described herein and apharmaceutically acceptable carrier or excipient.

In some embodiments, the MEK inhibitor is a competitive inhibitor ofMEK. In some embodiments, the MEK inhibitor is more selective against anactivating KRAS mutation. In some embodiments, the MEK inhibitor is anallosteric inhibitor of MEK. In some embodiments, the MEK inhibitor ismore selective against an activating BRAF mutation (e.g., BRAF V600Emutation). In some embodiments, the MEK inhibitor binds and inhibits theactivity of MEK1 and/or MEK2 (such as human MEK I and/or human MEK2).

In some embodiments, the MEK inhibitor is a compound selected from thegroup consisting of GDC-0973, G-38963, G02443714 (also known as“AS703206”), G02442104 (also known as “GSK-1120212”), and G00039805(also known as “AZD-6244”), or a pharmaceutically acceptable salt orsolvate thereof.

In some embodiments, the MEK inhibitor is a compound of formula (I),

or a pharmaceutically acceptable salt or solvate thereof, wherein A, X,R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are as defined in Group A, Group B, GroupC, or Group D:

Group A:

-   A is arylene optionally substituted with one, two, three or four    groups selected from R¹⁰, R¹², R¹⁴, R¹⁶, and R¹⁹ where R¹⁰, R¹², R¹⁴    and R¹⁶ are independently hydrogen, alkyl, alkenyl, alkynyl, halo,    haloalkoxy, hydroxy, alkoxy, amino, alkylamino, dialkylamino,    haloalkyl, —NHS(O)₂R⁸, —CN, —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′) and    —NR⁸C(O)R^(8′) and where R¹⁹ is hydrogen, alkyl, or alkenyl;-   X is alkyl, halo, haloalkyl, or haloalkoxy;-   R¹, R², R³, R⁴, R⁵ and R⁶ are independently hydrogen, halo, nitro,    —NR⁸R⁸, —OR⁸, —NHS(O)₂R⁸, —CN, —S(O)_(m)R⁸, —S(O)₂NR⁸R^(8′),    —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′), —NR⁸C(O)OR^(8′),    —NR⁸C(O)NR^(8′)R^(8″), NR⁸C(O)OR^(8′), —NR⁸C(O)R^(8′),    —CH₂N(R²⁵)(NR^(25a)R^(25b)), —CH₂NR²⁵C(═NH)(N^(25a)R^(25b)),    CH₂NR²⁵C(═NH)(N(R^(25a))(NO₂)), —CH₂NR²⁵C(═NH)(N(R^(25a))(CN)),    —CH₂NR²⁵C(═NH)(R²⁵), —CH₂NR²⁵C(NR^(25a)R^(25b))═CH(NO₂), alkyl,    alkenyl, alkynyl, cycloalkyl, heteroaryl, or heterocycloalkyl; where    the alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, and    heterocycloalkyl are independently optionally substituted with one,    two, three, four, five, six or seven groups independently selected    from halo, alkyl, haloalkyl, nitro, optionally substituted    cycloalkyl, optionally substituted heterocycloalkyl, optionally    substituted aryl, optionally substituted arylalkyl, optionally    substituted heteroaryl, —OR⁸, —NR⁸R^(8′), —NR⁸S(O)₂R⁹, —CN,    —S(O)_(m)R⁹, —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′),    NR⁸C(O)NR^(8′)R^(8″), —NR⁸C(O)OR^(8′) and —NR⁸C(O)R^(8′); or one of    R¹ and R² together with the carbon to which they are attached, R³    and R⁴ together with the carbon to which they are attached, and R⁵    and R⁶ together with the carbon to which they are attached form C(O)    or C(═NOH);-   m is 0, 1, or 2;-   R⁷ is hydrogen, halo or alkyl;-   each R⁸, R^(8′) and R^(8″) is independently selected from hydrogen,    hydroxy, optionally substituted alkoxy, alkyl, alkenyl, alkynyl,    aryl, cycloalkyl, heteroaryl, and heterocycloalkyl; where the alkyl,    alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl    are independently optionally substituted with one, two three, four,    or five groups independently selected from alkyl, halo, hydroxy,    hydroxyalkyl, optionally substituted alkoxy, alkoxyalkyl, haloalkyl,    carboxy, alkoxycarbonyl, alkenyloxycarbonyl, optionally substituted    cycloalkyl, optionally substituted cycloalkyloxycarbonyl, optionally    substituted aryl, optionally substituted aryloxy, optionally    substituted aryloxycarbonyl, optionally substituted arylalkyl,    optionally substituted arylalkyloxy, optionally substituted    arylalkyloxycarbonyl, nitro, cyano, optionally substituted    heterocycloalkyl, optionally substituted heteroaryl, —S(O)R³¹ (where    n is 0, 1, or 2 and R³¹ is optionally substituted alkyl, optionally    substituted aryl, optionally substituted heterocycloalkyl, or    optionally substituted heteroaryl), —NR³⁴SO₂R^(34a) (where R³⁴ is    hydrogen or alkyl and R³″ is alkyl, alkenyl, cycloalkyl, aryl,    heteroaryl, or heterocycloalkyl), —SO₂NR³⁵R^(35a) (where R³⁵ is    hydrogen or alkyl and R^(35a) is alkyl, alkenyl, cycloalkyl, aryl,    heteroaryl, or heterocycloalkyl), —NR³²C(O)R^(32a) (where R³² is    hydrogen or alkyl and R^(32a) is alkyl, alkenyl, alkoxy, or    cycloalkyl), —NR³⁰R^(30′) (where R³⁰ and R^(30′) are independently    hydrogen, alkyl, or hydroxyalkyl), and —C(O)NR³³R^(33a) (where R³³    is hydrogen or alkyl and R^(33a) is alkyl, alkenyl, alkynyl, or    cycloalkyl); and-   each R⁹ is independently selected from alkyl, alkenyl, alkynyl,    aryl, cycloalkyl, heteroaryl, and heterocycloalkyl; where the alkyl,    alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl    are independently optionally susbstituted with one, two, three,    four, or five groups selected from halo, hydroxy, alkyl, haloalkyl,    haloalkoxy, amino, alkylamino, and dialkylamino;

Group B:

-   A is heteroarylene optionally substituted with one, two, three, or    four groups selected from R¹⁰, R¹², R¹⁴, R¹⁶ and R¹⁹ where R¹⁰, R¹²,    R¹⁴ and R¹⁶ are independently hydrogen, alkyl, alkenyl, alkynyl,    halo, haloalkoxy, hydroxy, alkoxy, cyano, amino, alkylamino,    dialkylamino, haloalkyl, alkylsulfonylamino, alkylcarbonyl,    alkenylcarbonyl, alkoxycarbonyl, alkenyloxycarbonyl, aminocarbonyl,    alkylaminocarbonyl, dialkylaminocarbonyl, or alkylcarbonylamino;    where R¹⁹ is hydrogen, alkyl, or alkenyl; and where each alkyl and    alkenyl, either alone or as part of another group within R¹⁰, R¹²,    R¹⁴, R¹⁶, and R¹⁹, is independently optionally substituted with    halo, hydroxy, or alkoxy;-   X is alkyl, halo, haloalkyl, or haloalkoxy;-   R¹, R², R³, R⁴, R³ and R⁶ are independently hydrogen, halo, nitro,    —NR⁸R^(8′), —OR⁸, —NHS(O)₂R⁸, —CN, —S(O)_(m)R⁸, —S(O)₂NR⁸R^(8′),    —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′), —NR⁸C(O)OR^(8′),    —NR⁸C(O)NR^(8′)R^(8″), —NR⁸C(O)OR^(8′), —NR⁸C(O)R^(8′),    —CH₂N(R²⁵)(NR^(25a)R^(25b)), —CH₂NR²⁵C(═NH)(NR^(25a)R^(25b)),    —CH₂NR²⁵C(═NH)N(R^(25a))(NO₂)), —CH₂NR²⁵C(NH)(NR^(25a))(CN)),    —CH₂NR²⁵C(═NH)(R²⁵), —CH₂NR²⁵C(NR^(25a)N^(25b))═CH(NO₂), alkyl,    alkenyl, alkynyl, cycloalkyl, heteroaryl, or heterocycloalkyl, where    the alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, and    heterocycloalkyl are independently optionally substituted with one,    two, three, four, five, six or seven groups independently selected    from halo, alkyl, haloalkyl, nitro, optionally substituted    cycloalkyl, optionally substituted heterocycloalkyl, optionally    substituted aryl, optionally substituted arylalkyl, optionally    substituted heteroaryl, —OR⁸, —NR⁸R^(8′), —NR⁸S(O)₂R⁹, —CN,    —S(O)_(m)R⁹, —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′),    —NR⁸C(O)NR^(8′)R^(8″), —NR⁸C(O)OR^(8′) and —NR⁸C(O)R^(8′); or one of    R¹ and R² together with the carbon to which they are attached, R³    and R⁴ together with the carbon to which they are attached, and R⁵    and R⁶ together with the carbon to which they are attached form C(O)    or C(═NOH);-   m is 1 or 2;-   R⁷ is hydrogen, halo or alkyl; and-   each R⁸, R^(8′) and R^(8″) is independently selected from hydrogen,    hydroxy, optionally substituted alkoxy, alkyl, haloalkyl, alkenyl,    alkynyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, where    the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and    heterocycloalkyl are independently optionally substituted with one,    two three, four, or five groups independently selected from alkyl,    halo, hydroxy, hydroxyalkyl, optionally substituted alkoxy,    alkoxyalkyl, haloalkyl, carboxy, carboxy ester, nitro, cyano,    —S(O)_(n)R³¹ (where n is 0, 1, or 2 and R³¹ is optionally    substituted alkyl, optionally substituted aryl, optionally    substituted cycloalkyl, optionally substituted heterocycloalkyl, or    optionally substituted heteroaryl), —NR³⁶S(O)₂R^(36a) (where R³⁶ is    hydrogen, alkyl, or alkenyl and R^(36a) is alkyl, alkenyl,    optionally substituted aryl, optionally substituted cycloalkyl,    optionally substituted heterocycloalkyl, or optionally substituted    heteroaryl), —S(O)₂NR³⁷R^(37a) (where R³⁷ is hydrogen, alkyl, or    alkenyl and R^(37a) is alkyl, alkenyl, optionally substituted aryl,    optionally substituted cycloalkyl, optionally substituted    heterocycloalkyl, or optionally substituted heteroaryl), optionally    substituted cycloalkyl, optionally substituted heterocycloalkyl,    optionally substituted aryl; optionally substituted arylalkyl,    optionally substituted aryloxy, optionally substituted arylalkyloxy,    optionally substituted heteroaryl, —NHC(O)R³² (where R³² is alkyl,    alkenyl, alkoxy, or cycloalkyl) and —NR³⁰R^(30′) (where R³⁰ and    R^(30′) are independently hydrogen, alkyl, or hydroxyalkyl), and    —C(O)NHR³³ (where R³³ is alkyl, alkenyl, alkynyl, or cycloalkyl);

Group C: A is

-   where R¹⁰ is hydrogen, alkyl, alkenyl, alkynyl, halo, haloalkoxy,    hydroxy, alkoxy, amino, alkylamino, dialkylamino, haloalkyl,    —NHS(O)₂R⁸, —CN, —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′) and    —NR⁸C(O)R^(8′);-   R^(10a) is hydrogen, alkyl, or alkenyl;-   Y¹ is ═CH— or ═N—;-   X is alkyl, halo, haloalkyl, or haloalkoxy;-   R¹, R², R³, R⁴, R⁵ and R⁶ are independently hydrogen, halo, nitro,    —NR⁸R^(8′), —OR⁸, —NHS(O)₂R⁸, —CN, —S(O)_(m)R⁸, —S(O)₂NR⁸R^(8′),    —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′), —NR⁸C(O)OR^(8′),    —NR⁸C(O)NR^(8′)R^(8″), —NR⁸C(O)OR^(8′), —NR⁸C(O)R^(8′),    —CH₂N(R²⁵)NR^(25a)R^(25b)), —CH₂NR²⁵C(═NH)(NR^(25a)R^(25b)),    —CH₂NR²⁵C(═NH(N(R^(25a))(NO₂)), —CH₂NR²⁵C(═NH)(N(R^(25a))(CN)),    —CH₂NR²⁵C(═NH)(R²⁵), —CH₂NR²⁵C(NR^(25a)R^(25b))═CH(NO₂), alkyl,    alkenyl, alkynyl, cycloalkyl, heteroaryl, or heterocycloalkyl, where    the alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, and    heterocycloalkyl are independently optionally substituted with one,    two, three, four, five, six or seven groups independently selected    from halo, alkyl, haloalkyl, nitro, optionally substituted    cycloalkyl, optionally substituted heterocycloalkyl, optionally    substituted aryl, optionally substituted arylalkyl, optionally    substituted heteroaryl, —OR⁸, —NR⁸R^(8′), —NR⁸S(O)₂R⁹, —CN,    —S(O)_(m)R⁹, —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′),    NR⁸C(O)NR^(8′)R^(8″), —NR⁸C(O)OR^(8′) and —NR⁸C(O)R^(8′); or one of    R¹ and R² together with the carbon to which they are attached, R³    and R⁴ together with the carbon to which they are attached, and R⁵    and R⁶ together with the carbon to which they are attached form C(O)    or C(NOH);-   m is 1 or 2;-   R⁷ is hydrogen, halo or alkyl; and-   each R⁸, R^(8′) and R^(8″) is independently selected from hydrogen,    hydroxy, optionally substituted alkoxy, alkyl, haloalkyl, alkenyl,    alkynyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, where    the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and    heterocycloalkyl are independently optionally substituted with one,    two three, four, or five groups independently selected from alkyl,    halo, hydroxy, hydroxyalkyl, optionally substituted alkoxy,    alkoxyalkyl, haloalkyl, carboxy, carboxy ester, nitro, cyano,    —S(O)_(n)R³¹ (where n is 0, 1, or 2 and R³¹ is optionally    substituted alkyl, optionally substituted aryl, optionally    substituted cycloalkyl, optionally substituted heterocycloalkyl, or    optionally substituted heteroaryl), —NR³⁶S(O)₂R^(36a) (where R³⁶ is    hydrogen, alkyl, or alkenyl and R^(36a) is alkyl, alkenyl,    optionally substituted aryl, optionally substituted cycloalkyl,    optionally substituted heterocycloalkyl, or optionally substituted    heteroaryl), —S(O)₂NR³⁷R^(37a) (where R³⁷ is hydrogen, alkyl, or    alkenyl and R^(37a) is alkyl, alkenyl, optionally substituted aryl,    optionally substituted cycloalkyl, optionally substituted    heterocycloalkyl, or optionally substituted heteroaryl), optionally    substituted cycloalkyl, optionally substituted heterocycloalkyl,    optionally substituted aryl, optionally substituted arylalkyl,    optionally substituted aryloxy, optionally substituted arylalkyloxy,    optionally substituted heteroaryl, —NHC(O)R³² (where R³² is alkyl,    alkenyl, alkoxy, or cycloalkyl) and —NR³⁰R^(30′) (where R³⁰ and    R^(30′) are independently hydrogen, alkyl, or hydroxyalkyl), and    —C(O)NHR³³ (where R³³ is alkyl, alkenyl, alkynyl, or cycloalkyl); or

Group D: A is

-   R⁴⁰ and R^(40a) are independently hydrogen or alkyl;-   X is alkyl, halo, haloalkyl, or haloalkoxy;-   R¹, R², R³, R⁴, R⁵ and R⁶ are independently hydrogen, halo, nitro,    —NR⁸R^(8′), —OR⁸, —NHS(O)₂R⁸, —CN, —S(O)_(m)R⁸, —S(O)₂NR⁸R⁸,    —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′), —NR⁸C(O)OR^(8′),    —NR⁸C(O)NR^(8′)R^(8″), —NR⁸C(O)OR^(8′), —NR⁸C(O)R^(8′),    —CH₂N(R²⁵)(NR^(25a)R^(25b)), —CH₂NR²⁵C(═NH)(NR^(25a)R^(25b)),    —CH₂NR²⁵C(═NH)(N(R^(25a)) (NO₂)), —CH₂NR²⁵C(═NH)(N(R^(25a))(CN)),    —CH₂NR²⁵C(═NH)(R²⁵), —CH₂NR²⁵C(NR^(25a)R^(25b))═CH(NO₂), alkyl,    alkenyl, alkynyl, cycloalkyl, heteroaryl, or heterocycloalkyl, where    the alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, and    heterocycloalkyl are independently optionally substituted with one,    two, three, four, five, six or seven groups independently selected    from halo, alkyl, haloalkyl, nitro, optionally substituted    cycloalkyl, optionally substituted heterocycloalkyl, optionally    substituted aryl, optionally substituted arylalkyl, optionally    substituted heteroaryl, —OR⁸, —NR⁸R^(8′), —NR⁸S(O)₂R⁹, —CN,    —S(O)_(m)R⁹, —C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R^(8′),    —NR⁸C(O)NR^(8′)R^(8″), —NR⁸C(O)OR^(8′) and —NR⁸C(O)R^(8′); or one of    R¹ and R² together with the carbon to which they are attached, R³    and R⁴ together with the carbon to which they are attached, and R⁵    and R⁶ together with the carbon to which they are attached form C(O)    or C(NOH);-   m is 1 or 2;-   R⁷ is hydrogen, halo or alkyl; and-   R⁸, R^(8′) and R^(8″) are independently selected from hydrogen,    hydroxy, optionally substituted alkoxy, alkyl, haloalkyl, alkenyl,    alkynyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, where    the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and    heterocycloalkyl are independently optionally substituted with one,    two three, four, or five groups independently selected from alkyl,    halo, hydroxy, hydroxyalkyl, optionally substituted alkoxy,    alkoxyalkyl, haloalkyl, carboxy, carboxy ester, nitro, cyano,    —S(O)_(n)R³¹ (where n is 0, 1, or 2 and R³¹ is optionally    substituted alkyl, optionally substituted aryl, optionally    substituted cycloalkyl, optionally substituted heterocycloalkyl, or    optionally substituted heteroaryl), —NR³⁶S(O)₂R^(36a) (where R³⁶ is    hydrogen, alkyl, or alkenyl and R^(36a) is alkyl, alkenyl,    optionally substituted aryl, optionally substituted cycloalkyl,    optionally substituted heterocycloalkyl, or optionally substituted    heteroaryl), —S(O)₂NR³⁷R^(37a) (where R³⁷ is hydrogen, alkyl, or    alkenyl and R^(37a) is alkyl, alkenyl, optionally substituted aryl,    optionally substituted cycloalkyl, optionally substituted    heterocycloalkyl, or optionally substituted heteroaryl), optionally    substituted cycloalkyl, optionally substituted heterocycloalkyl,    optionally substituted aryl, optionally substituted arylalkyl,    optionally substituted aryloxy, optionally substituted arylalkyloxy,    optionally substituted heteroaryl, —NHC(O)R³² (where R³² is alkyl,    alkenyl, alkoxy, or cycloalkyl) and —NR³⁰R^(30′) (where R³⁰ and    R^(30′) are independently hydrogen, alkyl, or hydroxyalkyl), and    —C(O)NHR³³ (where R³³ is alkyl, alkenyl, alkynyl, or cycloalkyl).

In some variations, the MEK inhibitor compound of the formula (I) is acompound of the Group A, having the formula I(a) or I(b):

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined for the formula (I), Group A, or as defined inWO 2007/044515 A1, incorporated herein by reference.

In some variations, the MEK inhibitor compound of the formula (I) is acompound of the Group B, having the formula I(c), I(d), I(e), I(f),I(g), I(h), I(i), I(j), I(k), I(m), I(n), I(O), I(p), I(q), I(r), I(s),I(u), I(v), I(w), I(x), I(cc) or I(dd):

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined for the formula (I), Group B, or as defined inWO 2007/044515 A1, incorporated herein by reference.

In some variations, the MEK inhibitor compound of the formula (I) is acompound of the Group C, having the formula I(y) or I(z):

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined for the formula (I), Group C, or as defined inWO 2007/044515 A1, incorporated herein by reference.

In some variations, the MEK inhibitor compound of the formula (I) is acompound of the Group D, having the formula I(aa) or I(bb):

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined for the formula (I), Group D, or as defined inWO 2007/044515 A1, incorporated herein by reference.

In some embodiments, the MEK inhibitor compound of the formula (I) is acompound selected from the compound Nos. 1-362 as listed in WO2007/044515 A1, Table 1 on pages 71-144 (herein collectively referred toas the Formula I Species), or a pharmaceutically acceptable salt orsolvate thereof.

Also embraced are any variations of formula (I) as described in WO2007/044515 A1, which is incorporated herein by reference. Compounds ofthe formula (I) or any variations thereof can be synthesized usingmethods known in the art, for example, the synthetic methods describedin WO 2007/044515 A1, incorporated herein by reference.

Unless defined otherwise herein, the terms used in describing compoundsof the formula (I) should be understood to have the same meaning asdefined in WO 2007/044515 A1.

In some embodiments, the MEK inhibitor is a compound of formula (II):

or a pharmaceutically acceptable salt or solvate thereof, wherein:

Z¹ is CR¹ or N;

Z² is CR² or N;

Z³ is CR³ or N;

Z⁴ is CR⁴ or N;

where one or two of Z¹, Z², Z³, and Z⁴ are N;

R¹, R², R³ and R⁴ are independently selected from H, halo, CN, CF₃,—OCF₃, —NO₂, —(CR¹⁴R¹⁵)C(═Y)R¹¹, —(CR¹⁴R¹⁵)_(n)C(═Y)OR¹¹,—(CR¹⁴R¹⁵)_(n)C(═Y)NR¹¹R¹², —(CR¹⁴R¹⁵)_(n)NR¹¹R¹², —(CR¹⁴R¹⁵)_(n)OR¹¹,—(CR¹⁴R¹⁵)_(n)SR¹¹, —(CR¹⁴R¹⁵)_(n)NR¹²C(═Y)R¹¹,—(CR¹⁴R¹⁵)_(n)NR¹²C(═Y)OR¹¹, —(CR¹⁴R¹⁵)_(n)NR¹³C(═Y)NR¹¹R¹²,—(CR¹⁴R¹⁵)_(n)NR¹²SO₂R¹¹, —(CR¹⁴R¹⁵)_(n)OC(═Y)R¹¹,—(CR¹⁴R¹⁵)_(n)OC(═Y)OR¹¹, —(CR¹⁴R¹⁵)_(n)OC(═Y)NR¹¹R¹²,—(CR¹⁴R¹⁵)OS(O)₂(OR¹¹), —(CR¹⁴R¹⁵)_(n)OP(═Y)(OR¹¹)(OR¹²),—(CR¹⁴R¹⁵)_(n)OP(OR¹¹)(OR¹²), —(CR¹⁴R¹⁵)_(n)S(O)R¹¹,—(CR¹⁴R¹⁵)_(n)S(O)₂R¹¹, —(CR¹⁴R¹⁵)_(n)S(O)₂NR¹¹R¹²,—(CR¹⁴R¹⁵)_(n)S(O)(OR¹¹), —(CR¹⁴R¹⁵)_(n)S(O)₂(OR¹¹),—(CR¹⁴R¹⁵)_(n)SC(═Y)R¹¹,—(CR¹⁴R¹⁵)_(n)SC(═Y)OR¹¹—(CR¹⁴R¹⁵)_(n)SC(═Y)NR¹¹R¹², C₁-C₁₂ alkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl;

W is

R⁵ and R⁶ are independently selected from H or C, —C₁₋₂ alkyl;

X¹ is selected from R¹¹, —OR¹¹, —NR¹¹R¹², —S(O)R¹¹, and —S(O)₂R¹¹; whenX¹ is R¹¹ or —OR¹¹, R¹¹ or —OR¹¹ of X¹ and —R⁵ are optionally takentogether with the nitrogen atom to which they are attached to form a 4-7membered saturated or unsaturated ring having 0-2 additional heteroatomsselected from O, S and N, wherein said ring is optionally substitutedwith one or more groups selected from halo, CN, CF₃, —OCF₃, —NO₂, oxo,—Si(C₁-C₆ alkyl), —(CR¹⁹R²⁰)_(n)C(═Y′)R¹⁶, —(CR¹⁹R²⁰)_(n)C(═Y′)OR¹⁶,—(CR¹⁹R²⁰)_(n)C(═Y′)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)OR⁶,—(CR¹⁹R²⁰)_(n)—SR¹⁶, —(CR¹⁹R²⁰)_(n)NR¹⁶C(═Y′)R¹⁷,—(CR¹⁹R²⁰)_(n)NR¹⁶C(═Y′)OR¹⁷, —(CR¹⁹R²⁰)_(n)NR¹⁸C(═Y′)NR¹⁶R¹⁷,—(CR¹⁹R²⁰)_(n)NR¹⁷SO₂R¹⁶, —(CR¹⁹R²⁰)_(n)OC(—Y′)R¹⁶,—(CR¹⁹R²⁰)_(n)OC(—Y′)OR¹⁶, —(CR¹⁹R²⁰)_(n)OC(═Y′)NR¹⁶R¹⁷,—(CR¹⁹R²⁰)OS(O)₂(OR¹⁶), —(CR¹⁹R²⁰)_(n)OP(═Y′)(OR¹⁶)(OR¹⁷),—(CR¹⁹R²⁰)_(n)OP(OR¹⁶)(OR¹⁷), —(CR¹⁹R²⁰)_(n)S(O)R¹⁶,—(CR¹⁹R²⁰)_(n)S(O)R¹⁶, —(CR¹⁹R²⁰)_(n)S(O)₂NR¹⁶R¹⁷,—(CR¹⁹R²⁰)_(n)S(O)(OR¹⁶), —(CR¹⁹R²⁰)_(n)S(O)₂(OR¹⁶),—(CR¹⁹R²⁰)_(n)SC(═Y′)R¹⁶, —(CR¹⁹R²⁰)_(n)SC(═Y′)OR¹⁶,—(CR¹⁹R²⁰)_(n)SC(═Y′)NR¹⁶R¹⁷, and R²¹;

X² is selected from carbocyclyl, heterocyclyl, aryl, and heteroaryl;

R¹¹, R¹² and R¹³ are independently H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl,

or R¹¹ and R¹² together with the nitrogen to which they are attachedform a 3-8 membered saturated, unsaturated or aromatic ring having 0-2heteroatoms selected from O, S and N, wherein said ring is optionallysubstituted with one or more groups selected from halo, CN, CF₃, —OCF₃,—NO₂, C₁-C₆ alkyl, —OH, —SH, —O(C₁-C₆ alkyl), —S(C₁-C₆ alkyl), —NH₂,—NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —SO₂(C₁-C₆ alkyl), —CO₂H, —CO₂(C₁-C₆alkyl), —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)(C₁-C₆ alkyl), —NHC(O)(C₁-C₆ alkyl), —NHSO₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)SO₂(C₁-C₆ alkyl), —SO₂NH₂, —SO₂NH(C₁-C₆ alkyl),—SO₂N(C₁-C₆ alkyl)₂, —OC(O)NH₂, —OC(O)NH(C₁-C₆ alkyl), —OC(O)N(C₁-C₆alkyl)₂, —OC(O)O(C₁-C₆ alkyl), —NHC(O)NH(C₁-C₆ alkyl),—NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)C(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)NH(C₁-C₆ alkyl), —NHC(O)N(C₁-C₆alkyl)₂, —NHC(O)O(C₁-C₆ alkyl), and —N(C₁-C₆alkyl)C(O)O(C₁-C₆ alkyl);

R¹⁴ and R¹⁵ are independently selected from H, C₁-C₂ alkyl, aryl,carbocyclyl, heterocyclyl, and heteroaryl;

m and n are independently selected from 0, 1, 2, 3, 4, 5, or 6;

Y is independently O, NR¹¹, or S;

wherein each said alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl and heteroaryl of R¹, R², R³, R⁴, R⁵, R⁶, X¹, X², R¹¹, R¹², R¹³,R¹⁴, and R¹⁵ is independently optionally substituted with one or moregroups independently selected from halo, CN, CF₃, —OCF₃, —NO₂, oxo,—Si(C₁-C₆ alkyl), —(CR¹⁹R²⁰)_(n)C(═Y′)R¹⁶, —(CR¹⁹R²⁰)_(n)C(═Y′)OR¹⁶,—(CR¹⁹R²⁰)_(n)C(═Y′)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)OR¹⁶,—(CR¹⁹R²⁰)_(n)—SR¹⁶, —(CR¹⁹R²⁰)_(n)NR¹⁶C(═Y′)R¹⁷,—(CR¹⁹R²⁰)_(n)NR¹⁶C(═Y′)OR¹⁷, —(CR¹⁹R²⁰⁾ _(n)NR^(18l C(═Y′)NR) ¹⁶R¹⁷,—(CR¹⁹R²⁰)_(n)NR¹⁷SO₂R¹⁶, —(CR¹⁹R^(20m))_(n)OC(═Y′)R¹⁶,—(CR¹⁹R²⁰)_(n)OC(═Y′)OR¹⁶, —(CR¹⁹R²⁰)_(n)OC(═Y′)NR¹⁶R¹⁷,—(CR¹⁹R²⁰)_(n)OS(O)₂(OR¹⁶), —(CR¹⁹R²⁰)_(n)OP(═Y′)(OR¹⁶)(OR¹⁷),—(CR¹⁹R²⁰)_(n)OP(OR¹⁶)(OR¹⁷), —(CR¹⁹R²⁰)_(n)S(O)R¹⁶,—(CR¹⁹R²⁰)_(n)S(O)₂R¹⁶, —(CR¹⁹R²⁰)_(n)S(O)₂NR¹⁶R¹⁷,—(CR¹⁹R¹⁷)_(n)S(O)(OR¹⁶), —(CR¹⁹R²⁰)_(n)S(O)₂(OR¹⁶), —(CR¹⁹R²⁰)_(n)SC(═Y′)R¹⁶, —(CR¹⁹R²⁰)_(n)SC(═Y′)OR¹⁶, —(CR¹⁹R²⁰)_(n)SC(═Y′)NR¹⁶R¹⁷, andR²¹;

each R¹⁶, R¹⁷ and R¹⁸ is independently H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, whereinsaid alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with one or more groups selectedfrom halo, oxo, CN, —OCF₃, CF₃, —NO₂, C₁-C₆ alkyl, —OH, —SH, —O(C₁-C₆alkyl), —S(C₁-C₆ alkyl), —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂,—SO₂(C₁-C₆ alkyl), —CO₂H, —CO₂(C₁-C₆ alkyl), —C(O)NH₂, —C(O)NH(C₁-C₆alkyl), —C(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)(C₁-C₆ alkyl),—NHC(O)(C₁-C₆ alkyl), —NHSO₂(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)SO₂(C₁-C₆alkyl), —SO₂NH₂, —SO₂NH(C₁-C₆ alkyl), —SO₂N(C₁-C₆ alkyl)₂, —OC(O)NH₂,—OC(O)NH(C₁-C₆ alkyl), —OC(O)N(C₁-C₆ alkyl)₂, —OC(O)O(C₁-C₆ alkyl),—NHC(O)NH(C₁-C₆ alkyl), —NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)C(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,—NHC(O)NH(C₁-C₆ alkyl), —NHC(O)N(C₁-C₆ alkyl)₂, —NHC(O)O(C₁-C₆ alkyl),and —N(C₁-C₆alkyl)C(O)O(C₁-C₆ alkyl);

or R¹⁶ and R¹⁷ together with the nitrogen to which they are attachedform a 3-8 membered saturated, unsaturated or aromatic ring having 0-2heteroatoms selected from O, S and N, wherein said ring is optionallysubstituted with one or more groups selected from halo, CN, —OCF₃, CF₃,—NO₂, C₁-C₆ alkyl, —OH, —SH, —O(C₁-C₆ alkyl), —S(C₁-C₆ alkyl), —NH₂,—NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —SO₂(C₁-C₆ alkyl), —CO₂H, —CO₂(C₁-C₆alkyl), —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)(C₁-C₆ alkyl), —NHC(O)(C₁-C₆ alkyl), —NHSO₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)SO₂(C₁-C₆ alkyl), —SO₂NH₂, —SO₂NH(C₁-C₆ alkyl),—SO₂N(C₁-C₆ alkyl)₂, —OC(O)NH₂, —OC(O)NH(C₁-C₆ alkyl), —OC(O)N(C₁-C₆alkyl)₂, —OC(O)O(C₁-C₆ alkyl), —NHC(O)NH(C₁-C₆ alkyl),—NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)C(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)NH(C₁-C₆ alkyl), —NHC(O)N(C₁-C₆alkyl)₂, —NHC(O)O(C₁-C₆ alkyl), and —N(C₁-C₆alkyl)C(O)O(C₁-C₆ alkyl);

R¹⁹ and R²⁰ are independently selected from H, C₁-C₁₂ alkyl,—(CH₂)_(n)-aryl, —(CH₂)_(n)-carbocyclyl, —(CH₂)_(n)-heterocyclyl, and—(CH₂)_(n)-heteroaryl;

R²¹ is C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, carbocyclyl,heterocyclyl, aryl, or heteroaryl, wherein each member of R²¹ isoptionally substituted with one or more groups selected from halo, CN,—OCF₃, CF₃, —NO₂, C₁-C₆ alkyl, —OH, —SH, —O(C₁-C₆ alkyl),—S(C₁-C₆alkyl), —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —SO(C₁-C₆alkyl), —CO₂H, —CO₂(C₁-C₆alkyl), —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl),—C(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)(C₁-C₆alkyl), —NHC(O)(C₁-C₆alkyl), —NHSO₂(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)SO₂(C₁-C₆ alkyl), —SO₂NH₂,—SO₂NH(C₁-C₆ alkyl), —SO₂N(C₁-C₆ alkyl)₂, —OC(O)NH₂, —OC(O)NH(C₁-C₆alkyl), —OC(O)N(C₁-C₆ alkyl)₂, —OC(O)O(C₁-C₆ alkyl), —NHC(O)NH(C₁-C₆alkyl), —NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)C(O)NH(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)NH(C₁-C₆ alkyl),—NHC(O)N(C₁-C₆ alkyl)₂, —NHC(O)O(C₁-C₆ alkyl), and—N(C₁-C₆alkyl)C(O)O(C₁-C₆ alkyl);

each Y′ is independently O, NR²², or S; and

R²² is H or C₁-C₁₂ alkyl.

In some variations, the MEK inhibitor compound of the formula (II) is acompound of the formula (II-1-a), (II-1-b), (II-1-c), (II-1-d),(II-1-e), (II-1-f), (II-1-g), (II-1-h), (II-1-i), (II-2-a), (II-2-b),(II-2-c), (II-2-d), (II-2-c), (II-2-f), (II-2-g), (II-2-h), (II-2-i),(II-3-a), (II-3-b), (II-3-c), (II-3-d), (II-3-e), (II-3-f), (II-3-g),(II-3-h), or (II-3-i):

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined for the formula (II) or as defined in WO2008/024725 A1, incorporated herein by reference.

In some embodiments, the MEK inhibitor compound of the formula (II) is acompound selected from the compounds of Examples 5-18, 20-102, 105-109,111-118, 120-133, 136-149 and 151-160 in WO 2008/024725 A1 (hereincollectively referred to as the Formula II Species), or apharmaceutically acceptable salt or solvate thereof. These compoundsexhibited an IC₅₀ of less than 10 μM in the assay described either inExample 8a or 8b (MEK activity assays). Most of these compoundsexhibited an IC₅₀ of less than 5 μM. See page 62 in WO 2008/024725 A1.

Also embraced are MEK inhibitor compounds (and/or solvates and saltsthereof) described in WO 2008/024725 A1, which is incorporated herein byreference, for example, aza-benzofuran compounds of the formula (II)(designated as formula I in WO 2008/024725 A1, e.g., on page 3) andvariations thereof as described in WO 2008/024725 A1. Compounds offormula (II) can be synthesized using methods known in the art, forexample, the synthetic methods described in WO 2008/024725 A1,incorporated herein by reference.

In some embodiments, the MEK inhibitor is a compound of formula (III):

or a pharmaceutically acceptable salt or solvate thereof, wherein:

Z¹ is CR¹ or N;

R¹ is H, C₁-C₃ alkyl, halo, CF₃, CHF₂, CN, OR^(A) or NR^(A)R^(A);

R^(1′)is H, C₁-C₃ alkyl, halo, CF₃, CHF₂, CN, OR^(A), or NR^(A)R^(A);

wherein each R^(A) is independently H or C₁-C₃ alkyl;

Z² is CR² or N;

Z³ is CR³ or N; provided that only one of Z¹, Z² and Z³ can be N at thesame time;

R² and R³ are independently selected from H, halo, CN, CF₃, —OCF₃, —NO₂,—(CR¹⁴R¹⁵)_(n)C(═Y′)R¹¹, (CR¹⁴R¹⁵)_(n)C(═Y′)OR¹¹,—(CR¹⁴R¹⁵)_(n)C(═Y′)NR¹¹R¹², —(CR¹⁴R¹⁵)_(n)NR¹¹R¹², —(CR¹⁴R¹⁵)_(n)OR¹¹,—(CR¹⁴R¹⁵)_(n)SR¹¹—(CR¹⁴R¹⁵)_(n)NR¹²C(═Y′)R¹¹,—(CR¹⁴R¹⁵)_(n)NR¹²C(═Y′)OR¹¹, —(CR¹⁴R¹³)_(n)NR¹³C(═Y′)NR¹¹R¹²,—(CR¹⁴R¹⁵)_(n)NR¹²SO₂R¹¹, —(CR¹⁴R¹⁵)_(n)OC(—Y′)R¹¹,—(CR¹⁴R¹⁵)_(n)OC(═Y′)OR¹¹, —(CR¹⁴R¹⁵)_(n)OC(═Y′)NR¹¹R¹²,—(CR¹⁴R¹⁵)_(n)OS(O)₂(OR¹¹), —(CR¹⁴R¹⁵)_(n)OP(═Y′)(OR¹¹)(OR¹²),—(CR¹⁴R¹⁵)OP(OR¹¹)(OR¹²), —(CR¹⁴R¹⁵)_(n)S(O)R¹¹, —(CR¹⁴R¹⁵)_(n)S(O)₂R¹¹,—(CR¹⁴R¹⁵)_(n)S(O)₂NR¹¹R¹², —(CR¹⁴R¹⁵)_(n)S(O)(OR¹¹),—(CR¹⁴R¹⁵)_(n)S(O)₂(OR¹¹), —(CR¹⁴R¹⁵)_(n)SC(═Y′)R¹¹,—(CR¹⁴R¹⁵)_(n)SC(═Y′)OR¹¹, —(CR¹⁴R¹⁵)_(n)SC(═Y′)NR¹¹R¹², C₁-C₁₂ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl;

R⁴ is H, C₁-C₆ alkyl or C₃-C₄ carbocyclyl;

Y is W—C(O)— or W′;

W is or

R⁵ is H or C₁-C₁₂ alkyl;

X¹ is selected from R^(11′) and —OR^(11′); when X¹ is R^(11′), X¹ isoptionally taken together with R⁵ and the nitrogen atom to which theyare bound to form a 4-7 membered saturated or unsaturated ring having0-2 additional heteroatoms selected from O, S and N, wherein said ringis optionally substituted with one or more groups selected from halo,CN, CF₃, —OCF₃, —NO₂, oxo, —(CR¹⁹R²⁰)_(n)C(═Y′)R¹⁶,—(CR¹⁹R²⁰)_(n)C(═Y′)OR¹⁶, —(CR¹⁹R²⁰)_(n)C(═Y′)NR¹⁶R¹⁷,—(CR¹⁹R²⁰)_(n)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)OR⁶, —(CR¹⁹R²⁰)_(n)—SR¹⁶,—(CR¹⁹R²⁰)_(n)NR¹⁶C(═Y′)R¹⁷, —(CR¹⁹R²⁰)_(n)NR¹⁶C(═Y′)OR¹⁷,—(CR¹⁹R²⁰)_(n)NR¹⁸C(═Y′)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)NR¹⁷SO₂R¹⁶,—(CR¹⁹R²⁰)_(n)OC(═Y′)R¹⁶, —(CR¹⁹R²⁰)_(n)OC(═Y′)OR¹⁶,—(CR¹⁹R²⁰)_(n)OC(—Y′)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)OS(O)₂(OR¹⁶),—(CR¹⁹R²⁰)_(n)OP(Y′)(OR¹⁶)(OR¹⁷), —(CR¹⁹R²⁰)_(n)OP(OR¹⁶)(OR⁷),—(CR¹⁹R²⁰)_(n)S(O)R¹⁶, —(CR¹⁹R²⁰)_(n)S(O)₂R¹⁶,—(CR¹⁹R²⁰)_(n)S(O)₂NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)S(O)(OR¹⁶),—(CR¹⁹R²⁰)_(n)S(O)₂(OR¹⁶), —(CR¹⁹R²⁰)_(n)SC(═Y′)R¹⁶,—(CR¹⁹R²⁰)_(n)SC(═Y′)OR¹⁶, —(CR¹⁹R²⁰)_(n) SC(═Y′)NR¹⁶R¹⁷, and R²¹;

each R^(11′) is independently H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl;

R¹¹, R¹² and R¹³ are independently H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl,

or R¹¹ and R¹² together with the nitrogen to which they are attachedform a 3-8 membered saturated, unsaturated or aromatic ring having 0-2heteroatoms selected from O, S and N, wherein said ring is optionallysubstituted with one or more groups selected from halo, CN, CF₃, —OCF₃,—NO₂, C₁-C₆ alkyl, —OH, —SH, —O(C₁-C₆ alkyl), —S(C₁-C₆ alkyl), —NH₂,—NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —SO₂(C₁-C₆ alkyl), —CO₂H, —CO₂(C₁-C₆alkyl), —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)(C₁-C₆ alkyl), —NHC(O)(C₁-C₆ alkyl), —NHSO₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)SO₂(C₁-C₆ alkyl), —SO₂NH₂, —SO₂NH(C₁-C₆ alkyl),—SO₂N(C₁-C₆ alkyl)₂, —OC(O)NH₂, —OC(O)NH(C₁-C₆ alkyl), —OC(O)N(C₁-C₆alkyl)₂, —OC(O)O(C₁-C₆ alkyl), —NHC(O)NH(C₁-C₆ alkyl),—NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)C(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)NH(C₁-C₆ alkyl), —NHC(O)N(C₁-C₆alkyl)₂, —NHC(O)O(C₁-C₆ alkyl), and —N(C₁-C₆alkyl)C(O)O(C₁-C₆ alkyl);

R¹⁴ and R¹⁵ are independently selected from H, C₁-C₁₂ alkyl, aryl,carbocyclyl, heterocyclyl, and heteroaryl;

W is

wherein

is

each X² is independently O, S, or NR⁹;

each R⁷ is independently selected from H, halo, CN, CF₃, —OCF₃, —NO₂,—(CR¹⁴R¹⁵)_(n)C(═Y′)R¹¹, —(CR¹⁴R¹⁵)_(n)C(═Y′)OR¹¹,—(CR¹⁴R¹⁵)_(n)C(═Y′)NR¹¹R¹², —(CR¹⁴R¹⁵)_(n)NR¹¹R¹², —(CR¹⁴R¹⁵)_(n)OR¹¹,—(CR¹⁴R¹⁵)_(n)SR¹¹, —(CR¹⁴R¹⁵)_(n)NR¹²C(═Y′)R¹¹,—(CR¹⁴R¹⁵)_(n)NR¹²C(═Y′)OR¹¹, —(CR¹⁴R¹⁵)_(n)NR¹³C(—Y′)NR¹¹R¹²,—(CR¹⁴R¹⁵)_(n)NR¹²SO₂R¹¹, —(CR¹⁴R¹⁵)_(n)OC(═Y′)R¹¹,—(CR¹⁴R¹⁵)_(n)OC(═Y′)OR¹¹, —(CR¹⁴R¹⁵)_(n)OC(—Y′)NR¹¹R¹²,—(CR¹⁴R¹⁵)_(n)OS(O)₂(OR¹¹), —(CR¹⁴R¹⁵)_(n)OP(═Y′)(OR¹¹)(OR¹²),—(CR¹⁴R¹⁵)_(n)OP(OR¹¹)(OR¹²), —(CR¹⁴R¹⁵)S(O)R¹¹, —(CR¹⁴R¹⁵)_(n)S(O)₂R¹¹,—(CR¹⁴R¹⁵)_(n)S(O)₂NR¹¹R¹², —(CR¹⁴R¹⁵)_(n)S(O)(OR¹¹),—(CR¹⁴R¹⁵)_(n)S(O)₂(OR¹¹), —(CR¹⁴R¹⁵)_(n)SC(═Y′)R¹¹,—(CR¹⁴R¹⁵)_(n)SC(═Y′)OR¹¹, —(CR¹⁴R¹⁵)_(n)SC(═Y′)NR¹¹R¹², C₁-C₁₂ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl;

each R⁸ is independently selected from C₁-C₁₂ alkyl, aryl, carbocyclyl,heterocyclyl, and heteroaryl;

R⁹ is selected from H, —(CR¹⁴R¹⁵)_(n)C(—Y′)R¹¹,—(CR¹⁴R¹⁵)_(n)C(═Y′)OR¹¹, —(CR¹⁴R¹⁵)_(n)C(═Y′)NR¹¹R¹²,(CR¹⁴R¹⁵)_(q)NR¹¹R¹², —(CR¹⁴R¹⁵)_(q)OR¹¹, —(CR¹⁴R¹⁵)_(q)SR¹¹,—(CR¹⁴R¹⁵)_(q)NR¹²C(═Y′)R¹¹, —(CR¹⁴R¹⁵)_(q)NR¹²C(═Y′)OR¹¹,—(CR¹⁴R¹⁵)_(q)NR¹³C(═Y′)NR¹¹R¹², —(CR¹⁴R¹⁵)_(q)NR¹²SO₂R¹¹,—(CR¹⁴R¹⁵)_(q)OC(═Y′)R¹¹, —(CR¹⁴R¹⁵)_(q)OC(═Y′)OR¹¹,—(CR¹⁴R¹⁵)_(q)OC(═Y′)NR¹¹R¹², —(CR¹⁴R¹⁵)_(q)OS(O)₂(OR¹¹),—(CR¹⁴R¹⁵)_(q)OP(═Y′)(OR¹¹)(OR¹²), —(CR¹⁴R¹⁵)_(q)OP(OR¹¹)(OR¹²),—(CR¹⁴R¹⁵)_(n)S(O)R¹¹, —(CR¹⁴R¹⁵)_(n)S(O)₂R¹¹, —(CR¹⁴R¹⁵)_(n)S(O)₂NR¹¹R¹², C₁-C₁₂ alkyl, C₁-C₈ alkenyl, C₂-C₈ alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl;

R¹⁰ is H, C₁-C₆ alkyl or C₃-C₄ carbocyclyl;

X⁴ is

R⁶ is H, halo, C₁-C₆ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, carbocyclyl,heteroaryl, heterocyclyl, —OCF₃, —NO₂, —Si(C₁-C₆ alkyl),—(CR¹⁹R²⁰)_(n)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)OR¹⁶, or —(CR¹⁹R²⁰)_(n)—SR¹⁶;

R^(6′) is H, halo, C₁-C₆ alkyl, carbocyclyl, CF₃, —OCF₃, —NO₂, —Si(C₁-C₆alkyl), —(CR¹⁹R²⁰)_(n)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)OR¹⁶, —(CR¹⁹R²⁰)_(n)—SR¹⁶,C₂-C₈ alkenyl, C₂-C₈ alkynyl, heterocyclyl, aryl, or heteroaryl;

p is 0, 1, 2 or 3;

n is 0, 1, 2 or 3;

q is 2 or 3

wherein each said alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl and heteroaryl of R¹, R², R³, R⁴, R⁵, R⁶, R^(6′), R⁷, R⁸, R⁹, R¹⁰,R¹¹, R^(11′), R¹², R¹³, R¹⁴, R¹⁵ and R^(A) is independently optionallysubstituted with one or more groups independently selected from halo,CN, CF₃, —OCF₃, —NO₂, oxo, —Si(C₁-C₆ alkyl), —(CR¹⁹R²⁰)_(n)C(═Y′)R¹⁶,—(CR¹⁹R²⁰)_(n)C(—Y′)OR¹⁶, —(CR¹⁹R²⁰)_(n)C(═Y′)NR¹⁶R¹⁷,—(CR¹⁹R²⁰)_(n)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)OR¹⁶, —(CR¹⁹R²⁰)_(n)SR¹⁶,—(CR¹⁹R²⁰)_(n)NR¹⁶C(═Y′)R¹⁷, —(CR¹⁹R²⁰)_(n)NR¹⁶C(═Y′)OR¹⁷,—(CR¹⁹R²⁰)_(n)NR¹⁸C(═Y′)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)NR¹⁷SO₂R¹⁶,—(CR¹⁹R²⁰)_(n)OC(═Y′)R¹⁶, —(CR¹⁹R²⁰)_(n)OC(═Y′)OR¹⁶,—(CR¹⁹R²⁰)OC(═Y′)NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)OS(O)₂(OR¹⁶),—(CR¹⁹R²⁰)_(n)OP(═Y′)(OR¹⁶)(OR¹⁷), —(CR¹⁹R²⁰)_(n)OP(OR¹⁶)(OR¹⁷),—(CR¹⁹R²⁰)_(n)S(O)R¹⁶, —(CR¹⁹R²⁰)_(n)S(O)₂R¹⁶,—(CR¹⁹R²⁰)_(n)S(O)₂NR¹⁶R¹⁷, —(CR¹⁹R²⁰)_(n)S(O)(OR¹⁶),—(CR¹⁹R²⁰)_(n)S(O)₂(OR¹⁶), —(CR¹⁹R²⁰)SC(═Y′)R⁶,—(CR¹⁹R²⁰)_(n)SC(═Y′)OR¹⁶, —(CR¹⁹R²⁰)_(n)SC(═Y′)NR¹⁶R¹¹, and R²¹;

each R¹⁶, R¹⁷ and R¹⁸ is independently H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, whereinsaid alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with one or more groups selectedfrom halo, CN, —OCF₃, CF₃, —NO₂, C₁-C₆ alkyl, —OH, —SH, —O(C₁-C₆ alkyl),—S(C₁-C₆ alkyl), —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —SO₂(C₁-C₆alkyl), —CO₂H, —CO₂(C₁-C₆ alkyl), —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl),—C(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)(C₁-C₆ alkyl), —NHC(O)(C₁-C₆alkyl), —NHSO₂(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)SO₂(C₁-C₆ alkyl), —SO₂NH₂,—SO₂NH(C₁-C₆ alkyl), —SO₂N(C₁-C₆ alkyl)₂, —OC(O)NH₂, —OC(O)NH(C₁-C₆alkyl), —OC(O)N(C₁-C₆ alkyl)₂, —OC(O)O(C₁-C₆ alkyl), —NHC(O)NH(C₁-C₆alkyl), —NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)C(O)NH(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)NH(C₁-C₆ alkyl),—NHC(O)N(C₁-C₆ alkyl)₂, —NHC(O)O(C₁-C₆ alkyl), and—N(C₁-C₆alkyl)C(O)O(C₁-C₆ alkyl);

or R¹⁶ and R¹⁷ together with the nitrogen to which they are attachedform a 3-8 membered saturated, unsaturated or aromatic ring having 0-2heteroatoms selected from O, S and N, wherein said ring is optionallysubstituted with one or more groups selected from halo, CN, —OCF₃, CF₃,—NO₂, C₁-C₆ alkyl, —OH, —SH, —O(C₁-C₆ alkyl), —S(C₁-C₆ alkyl), —NH₂,—NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —SO₂(C₁-C₆ alkyl), —CO₂H, —CO₂(C₁-C₆alkyl), —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)(C₁-C₆ alkyl), —NHC(O)(C₁-C₆ alkyl), —NHSO₂(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)SO₂(C₁-C₆ alkyl), —SO₂NH₂, —SO₂NH(C₁-C₆ alkyl),—SO₂N(C₁-C₆ alkyl)₂, —OC(O)NH₂, —OC(O)NH(C₁-C₆ alkyl), —OC(O)N(C₁-C₆alkyl)₂, —OC(O)O(C₁-C₆ alkyl), —NHC(O)NH(C₁-C₆ alkyl),—NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)C(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)NH(C₁-C₆ alkyl), —NHC(O)N(C₁-C₆alkyl)₂, —NHC(O)O(C₁-C₆ alkyl), and —N(C₁-C₆alkyl)C(O)O(C₁-C₆ alkyl);

R¹⁹ and R²⁰ are independently selected from H, C₁-C₁₂ alkyl,—(CH₂)_(n)-aryl, —(CH₂)_(n)-carbocyclyl, —(CH₂)_(n)-heterocyclyl, and—(CH₂)_(n)-heteroaryl;

R²¹ is C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, carbocyclyl,heterocyclyl, aryl, or heteroaryl, wherein each member of R²¹ isoptionally substituted with one or more groups selected from halo, oxo,CN, —OCF₃, CF₃, —NO₂, C₁-C₆ alkyl, —OH, —SH, —O(C₁-C₆ alkyl), —S(C₁-C₆alkyl), —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —SO₂(C₁-C₆ alkyl),—CO₂H, —CO₂(C₁-C₆ alkyl), —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)C(O)(C₁-C₆ alkyl), —NHC(O)(C₁-C₆ alkyl),—NHSO₂(C₁-C₆ alkyl), —N(C₁-C₆alkyl)SO₂(C₁-C₆ alkyl), —SO₂NH₂,—SO₂NH(C₁-C₆ alkyl), —SO₂N(C₁-C₆ alkyl)₂, —OC(O)NH₂, —OC(O)NH(C₁-C₆alkyl), —OC(O)N(C₁-C₆ alkyl)₂, —OC(O)O(C₁-C₆ alkyl),—NHC(O)NH(C₁-C₆alkyl), —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)C(O)NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)C(O)N(C₁-C₆ alkyl)₂,—NHC(O)NH(C₁-C₆ alkyl), —NHC(O)N(C₁-C₆ alkyl)₂, —NHC(O)O(C₁-C₆ alkyl),and —N(C₁-C₆ alkyl)C(O)O(C₁-C₆ alkyl);

each Y′ is independently O, NR²², or S; and

R²² is H or C₁-C₁₂ alkyl.

In some variations, the MEK inhibitor compound of the formula (III) hasthe formula (III-a) or (II-b):

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined for the formula (III) or as defined in WO2009/085983 A1, incorporated herein by reference.

In some embodiments, the MEK inhibitor compound of the formula (III) isa compound selected from the compounds listed in Table 1, or apharmaceutically acceptable salt or solvate thereof.

TABLE 1 Compound No. Chemical Name Structure (III)-5  5-(2-Fluoro-4-iodophenylamino)-imidazo[1,5- a]pyridine-6-carboxylic acid (2-hydroxyethoxy)-amide

(III)-6  5-(2-Fluoro-4-iodo- phenylamino)-imidazo[1,5-a]pyridine-6-carboxylic acid ((R)-2,3-dihydroxy-propoxy)- amide

(III)-7  5-(2-Fluoro-4-iodo- phenylamino)-imidazo[1,5-a]pyridine-6-carboxylic acid ((S)-2-hydroxy-propoxy)-amide

(III)-8  5-(4-Bromo-2- fluorophenylamino)- imidazo[1,5-a]pyridine-6-carboxylic acid (2- hydroxyethoxy)-amide

(III)-9  5-(4-Bromo-2-fluoro- phenylamino)-imidazo[1,5-a]pyridine-6-carboxylic acid ((S)-2-hydroxy-propoxy)-amide

(III)-10 5-(4-Bromo-2-fluoro- phenylamino)-8-fluoro-imidazo[1,5-a]pyridine-6- carboxylic acid ((S)-2-hydroxy- propoxy)-amide

(III)-11 8-Fluoro-5-(2-fluoro-4-iodo- phenylamino)-imidazo[1,5-a]pyridine-6-carboxylic acid (2- hydroxy-ethoxy)-amide

(III)-12 8-Fluoro-5-(2-fluoro-4-iodo- phenylamino)-imidazo[1,5-a]pyridine-6-carboxylic acid ((R)-2,3-dihydroxy-propoxy)- amide

(III)-13 8-Fluoro-5-(2-fluoro-4-iodo- phenylamino)-imidazo[1,5-a]pyridine-6-carboxylic acid ((S)-2-hydroxy-propoxy)-amide

(III)-14 5-(2-Fluoro-methanesulfanyl- phenylamino)-imidazo[1,5-a]pyridine-6-carboxylic acid (2- hydroxy-ethoxy)-amide

(III)-15 5-(2-Fluoro-4-iodo- phenylamino)-imidazo[1,5-a]pyrazine-6-carboxylic acid (2- hydroxy-ethoxy)-amide

(III)-16 5-(2-Fluoro-4-iodo- phenylamino)-imidazo[1,5-a]pyrazine-6-carboxylic acid ((S)-2-hydroxy-propoxy)-amide

(III)-17 5-(4-Cyclopropyl-2-fluoro- phenylamino)-imidazo[1,5-a]pyridine-6-carboxylic acid (2- hydroxy-ethoxy)-amide

(III)-18 (R)-N-(2,3-Dihydroxypropoxy)- 5-(2-fluoro-4-iodophenylamino)imidazo[1,5- a]pyrazine-6-carboxamide

(III)-19 N-Ethoxy-5-(2-fluoro-4- iodophenylamino)imidazo[1,5-a]pyrazine-6-carboxamide

(III)-20 N-(Cyclopropylmethoxy)-5-(2- fluoro-4-iodophenylamino)imidazo[1,5- a]pyrazine-6-carboxamide

(III)-21 5-(2-Fluoro-4- iodophenylamino)-N-methylimidazo[1,5-a]pyrazine- 6-carboxamide

(III)-22 5-(4-Bromo-2- fluorophenylamino)-N-(2-hydroxy-ethoxy)imidazo[1,5- a]pyrazine-6-carboxamide

(III)-23 (S)-5-(4-Bromo-2- fluorophenylamino)-N-(2-hydroxy-propoxy)imidazo[1,5- a]pyrazine-6-carboxamide

(III)-24 (R)-5-(4-Bromo-2- fluorophenylamino)-N-(2,3-dihydroxy-propoxy)imidazo[1,5- a]pyrazine-6-carboxamide

(III)-25 5-(4-Bromo-2- fluorophenylamino)-N- (cyclopropyl-methoxy)imidazo[1,5- a]pyrazine-6-carboxamide

Compounds in Table 1 correspond to Examples 5-25 in WO 2009/085983 A1.Compounds (III)-5-(III)-20 and (III)-22-(III)-24 exhibited an IC₅₀ ofless than 0.5 μM in the assay described in Example 8b (MEK activityassay). Some of these compounds exhibited an IC₅₀ of less than 0.1 μM.Compounds (III)-21 and (III)-25 exhibited an IC₃₀ of less than 10 μM.See page 49 in WO 2009/085983 A1.

Also embraced are MEK inhibitor compounds (and/or solvates and saltsthereof) described in WO 2009/085983 A1, which is incorporated herein byreference, for example, imidazopyridine compounds of the formula (III)(designated as formula I in WO 2009/085983 A1, e.g., on page 3) andvariations thereof as described in WO 2009/085983 A1. Compounds offormula (III) can be synthesized using methods known in the art, forexample, the synthetic methods described in WO 2009/085983 A1,incorporated herein by reference.

In some embodiments, the MEK inhibitor is a compound of formula (IV),

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined in WO 03/077914 A1 for the formula I on pages4-9 or any applicable variations described in WO 03/077914 A1,incorporated herein by reference.

In some variations, the MEK inhibitor compound of the formula (IV) is acompound of the formula (IV-a), (N-b), (N-c), or (IV-d):

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined in WO 03/077914 A1 for the formulae II, III,IIIa and IIIb, respectively on pages 10-13 or any applicable variationsdescribed in WO 03/077914 A1, incorporated herein by reference.

In some embodiments, the MEK inhibitor compound of the formula (IV) is acompound selected from the group consisting of:

-   7-Fluoro-6-(4-bromo-2-methyl-phenylamino)-3H-benzoimidazole-5-carboxylic    acid cyclopropylmethoxy-amide;-   6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3H-benzoimidazole-5-carboxylic    acid cyclopropylmethoxy-amide;-   6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic    acid (2-hydroxy-ethoxy)-amide;-   6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic    acid (2,3-dihydroxy-propoxy)-amide;-   6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylmethyl)-3H-benzoimidazole-5-carboxylic    acid (2-hydroxy-ethoxy)-amide;-   [6-(5-Amino-[1,3,4]oxadiazol-2-yl)-4-fluoro-H-benzoimidazol-5-yl]-(4-bromo-2-methyl-phenyl)-amine;-   1-[6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-5-yl]-2-hydroxy-ethanone;-   1-[6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3H-benzoimidazol-5-yl]-2-methoxy    ethanone;-   6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic    acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide;-   6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylmethyl)-3H-benzoimidazole-5-carboxylic    acid (2-hydroxy-ethoxy)-amide;-   6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3H-benzoimidazole-5-carboxylic    acid (2-hydroxy-ethoxy)-amide;-   6-(-Bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic    acid (2-hydroxy-ethoxy)-amide; and-   6-(2,4-Dichloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic    acid (2-hydroxy-ethoxy)-amide;

or a pharmaceutically acceptable salt or solvate thereof.

Also embraced are any variations of formula (IV) as described in WO03/077914 A1, which is incorporated herein by reference. Compounds ofthe formula (IV) or any variations thereof can be synthesized usingmethods known in the art, for example, the synthetic methods describedin WO 03/077914 A1, incorporated herein by reference.

In some embodiments, the MEK inhibitor is a compound of formula (V),

or a pharmaceutically acceptable salt or solvate thereof, wherein thevariables are as defined in WO 2005/121142 A1 for the formula [1] onpages 6-10 or any applicable variations described in WO 2005/121142 A1,incorporated herein by reference.

Also embraced are any variations of formula (V) as described in WO2005/121142 A1, such as the individual MEK inhibitor compounds describedin WO 2005/121142 A1, e.g., Examples 1-1 to 1-343 in Table 1, Examples2-1 and 2-2 in Table 2, Examples 3-1 to 3-9 in Table 3, Examples 4-1 to4-148 in Table 4. Compounds of the formula (V) or any variations thereofcan be synthesized using methods known in the art, for example, thesynthetic methods described in WO 2005/121142 A1, incorporated herein byreference.

In some embodiments, the MEK inhibitor is a compound of formula (VI),

or a pharmaceutically acceptable salt or ester thereof, wherein:

R1 is selected from the group consisting of bromo, iodo, ethynyl,cycloalkyl, alkoxy, azetidinyl, acetyl, heterocycyl, cyano,straight-chained alkyl and branched-chain alkyl;

R2 is selected from the group consisting of hydrogen, chlorine,fluorine, and alkyl;

R3 is selected from the group consisting of hydrogen, chlorine, andfluorine;

R4 is selected from the group consisting of hydrogen, optionallysubstituted aryl, alkyl, and cycloalkyl;

R5 is selected from the group consisting of hydrogen and

wherein R6 is selected from the group consisting of hydroxyl, alkoxy,cycloalkyl, optionally substituted alkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R7 and R8 are independently selected from the group consisting ofhydrogen and optionally substituted alkyl;

or R6 and R7 can together form a cycloalkyl group and R8 is hydrogen.

In some variations, the MEK inhibitor compound is of the formula (VI),or a pharmaceutically acceptable salt or ester thereof, wherein thevariables are as defined in WO 2007/096259 A1 for the formula I or anyapplicable variations described on pages 4-10 in WO 2007/096259 A1,incorporated herein by reference. Further embraced MEK inhibitors arecompounds described in Examples 1-182 in WO 2007/096259 A1, incorporatedherein by reference.

In some embodiments, the MEK inhibitor compound of the formula (VI) is acompound selected from the group consisting of:

-   (2S,3S)—N-(4-Bromo-phenyl)-2-[(R)-4-(4-methoxy-phenyl)-2,5-dioxo-imidazolidin-1-yl]-3-phenyl-butyramide;-   (2S,3S)—N-(4-Iodo-phenyl)-2-[(R)-4-(4-methoxy-phenyl)-2,5-dioxo-imidazolidin-1-yl]3-phenyl-butyramide;-   (2S,3S)—N-(2-Fluoro-4-iodo-phenyl)-2-{(R)-4-[4-(2-hydroxy-ethoxy)-phenyl]-2,5-dioxo-imidazolidin-1-yl}-3-phenyl-butyramide;-   (2S,3S)—N-(4-Ethynyl-2-fluoro-phenyl)-2-{(R)-4-[4-(2-hydroxy-ethoxy)-phenyl]-2,5-dioxo-imidazolidin-1-yl}-3-phenyl-butyramide;-   (2R,3S)—N-(4-Ethynyl-2-fluoro-phenyl)-2-{(R)-4-[4-(2-hydroxy-ethoxy)-phenyl]-2,5-dioxo-imidazolidini-1-yl}-3-phenyl-butyramide;-   (2S,3S)—N-(2-Chloro-4-iodo-phenyl)-2-{(R)-4-[4-(2-hydroxy-ethoxy)-phenyl]-2,5-dioxo-imidazolidin-1-yl}-3-phenyl-butyramide;-   (2S,3S)-2-{(R)-4-[4-(2-Hydroxy-ethoxy)-phenyl]-2,5-dioxo-imidazolidin-1-yl)}-N-(4-iodo-2-methyl-phenyl)-3-phenyl-butyramide;-   (2S,3S)—N-(2-Chloro-4-iodo-phenyl)-2-{(R)-4-[4-((R)-2,3-dihydroxy-propoxy)-phenyl]-2,5-dioxo-imidazolidin-1-yl}-3-phenyl-butyramide;-   (2S,3S)—N-(2-Chloro-4-iodo-phenyl)-2-{(R)-4-[4-((S)-2,3-di    hydroxy-propoxy)-phenyl]-2,5-dioxo-imidazolidin-1-yl}-3-phenyl-butyramide;-   (2S,3S)-2-{(R)-2,5-Dioxo-4-[4-(2-oxo-2-pyrrolidin-1-yl-ethoxy)-phenyl]-imidazolidin-1-yl}-N-(2-fluoro-4-iodo-phenyl)-3-phenyl-butyramide;-   (2S,3S)-2-((R)-2,5-Dioxo-4-thiophen-3-yl-imidazolidin-1-yl)-N-(4-iodo-phenyl)-3-phenyl-butyramide;-   (S)-2-[(R)-4-(2,3-Dihydro-benzo[1,4]dioxin-6-yl)-2,5-dioxo-imidazolidin-1-yl]-N-(2-fluoro-4-iodo-phenyl)-3-phenyl-propionamide;-   (S)-2-[(R)-4-(4-Acetylamino-phenyl)-2,5-dioxo-imidazolidin-1-yl]-N-(2-fluoro-4-iodo-phenyl)-3-phenyl-propionamide;-   (4-{(R)-1-[(1S,2S)-1-(2-Fluoro-4-iodo-phenylcarbamoyl)-2-phenyl-propyl]-2,5-dioxo-imidazolidin-4-yl}-phenoxymethyl)-phosphonic    acid dimethyl ester;-   (2S,3S)—N-(2-Fluoro-4-iodo-phenyl)-2-((R)-4-isopropyl-2,5-dioxo-imidazolidin-1-yl)-3-phenyl-butyramide;-   (S)—N-(2-Fluoro-4-iodo-phenyl)-2-{(R)-4-[4-(2-hydroxy-ethoxy)-phenyl]-2,5-dioxo-imidazolidin-1-yl}=3-methyl-butyramide;-   (S)—N-(2-Fluoro-4-iodo-phenyl)-2-[(R)-4-(4-methoxy-phenyl)-2,5-dioxo-imidazolidin-1-yl]-3-o-tolyl-propionamide;-   (S)—N-(2-Fluoro-4-iodo-phenyl)-2-[(R)-4-(4-methoxy-phenyl)-2,5-dioxo-imidazolidin-1-yl]-3-m-tolyl-propionamide;-   (S)—N-(2-Fluoro-4-iodo-phenyl)-2-[(R)-4-(4-methoxy-phenyl)-2,5-dioxo-imidazolidin-1-yl]-3-p-tolyl-propionamide;    and-   (S)—N-(4-Cyclopropyl-2-fluoro-phenyl)-3-(4-fluoro-phenyl)-2-{(R)-4-[4-(2-hydroxy-1-hydroxymethyl-ethoxy)-phenyl]-2,5-dioxo-imidazolidin-1-yl}-propionamide;

or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, the MEK inhibitor is a compound of formula (VII),

or a pharmaceutically acceptable salt or ester thereof, wherein:

R1 is selected from the group consisting of halogen, ethynyl, andcycloalkyl;

R2 is selected from the group consisting of hydrogen and CH(R3)(R4);

R3 is selected from the group consisting of lower alkyl, lower alkoxy,optionally substituted aryl, and optionally substituted heteroaryl;

R4 is selected from the group consisting of hydrogen and lower alkyl;

R5 is hydrogen or, taken together with R2 and the carbon to which R2 andR5 are attached, forms lower cycloalkyl; and

R6 is selected from the group consisting of hydrogen, lower alkyl, lowercycloalkyl, optionally substituted aryl, and optionally substitutedheteroaryl.

In some variations, the MEK inhibitor compound is of the formula (VI),or a pharmaceutically acceptable salt or ester thereof, wherein thevariables are as defined in WO 2009/021887 A1 for the formula I or anyapplicable variations described on pages 4-5 in WO 2009/021887 A1,incorporated herein by reference. Further embraced MEK inhibitors arecompounds described in Examples 1-21 in 2009/021887 A1, incorporatedherein by reference.

In some embodiments, the MEK inhibitor compound of the formula (VI) is acompound selected from the group consisting of:

-   (R)-5-[4-(2-Hydroxy-ethoxy)-phenyl]-3-[(S)—-(6-iodo-1H-benzoimidazol-2-yl)-2-phenyl-ethyl]-imidazolidine-2,4-dione;-   (R)-5-[4-(2-Hydroxy-ethoxy)-phenyl]-3-(5-iodo-1H-benzoimidazol-2-ylmethyl)-imidazolidine-2,4-dione;-   (R)-5-[4-(2-Hydroxy-ethoxy)-phenyl]-3-[(S)-1-(5-iodo-1H-benzoimidazol-2-yl)-2-methyl-propyl]-imidazolidine-2,4-dione;-   (R)-5-[4-(2-Hydroxy-ethoxy)-phenyl]-3-[(1R,2R)-1-(5-iodo-1H-benzoimidazol-2-yl)-2-methoxy-propyl]-imidazolidine-2,4-dione;-   3-[(S)-1-(5-Iodo-1H-benzoimidazol-2-yl)-2-phenyl-ethyl]-imidazolidine-2,4-dione;    compound with trifluoro-acetic acid;-   (R)-3-[(S)-2-(4-Fluoro-phenyl)-1-(5-iodo-1H-benzoimidazol-2-yl)-ethyl]-5-[4-(2-hydroxy-ethoxy)-phenyl]-imidazolidine-2,4-dione;-   (R)-5-[4-(2-Hydroxy-ethoxy)-phenyl]-3-[(S)-1-(5-iodo-1H-benzoimidazol-2-yl)-2-(4-methoxy-phenyl)-ethyl]-imidazolidine-2,4-dione;-   (R)-5-[4-(2-Hydroxy-ethoxy)-phenyl]-3-[(S)-1-(5-iodo-1H-benzoimidazol-2-yl)-2-thiophen-2-yl-ethyl]-imidazolidine-2,4-dione;-   (R)-3-[(1S,2S)-1-(6-Iodo-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-5-phenyl-imidazolidine-2,4-dione;-   (R)-3-[(1S,2S)-1-(6-Iodo-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-5-(4-methoxy-phenyl)-imidazolidine-2,4-dione;-   (R)-5-[4-(2-Hydroxy-ethoxy)-phenyl]-3-[(1S,2S)-1-(6-iodo-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-imidazolidine-2,4-dione;-   (R)-3-[(1S,2S)-1-(6-Iodo-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-5-[4-(2-methoxy-ethoxy)-phenyl]-imidazol    idine-2,4-dione;-   2-(4-{(R)-1-[(1S,2S)-1-(6-Iodo-1H-benazoimidazol-2-yl)-2-phenyl-propyl]-2,5-dioxo-imidazolidin-4-yl)}-phenoxy)-N,N-dimethyl-acetamide;-   N,N-Bis-(2-hydroxy-ethyl)-2-(4-{(R)-1-[(1S,2S)-1-(6-iodo-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-2,5-dioxo-imidazolidin-4-yl}-phenoxy)-acetamide;-   (R)-3-[(1S,2S)-1-(5-Iodo-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-5-isopropyl-imidazolidine-2,4-dione;-   (R)-5-Cyclohexyl-3-[(1S,2S)-1-(5-iodo-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-imidazolidine-2,4-dione;-   (R)-5-[4-(2-Hydroxy-ethoxy)-phenyl]-3-[1-(5-iodo-1H-benzoimidazol-2-yl)-cyclopropyl]-imidazolidine-2,4-dione;-   (R)-3-[(1S,2S)-1-(6-Bromo-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-5-[4-(2-hydroxy-ethoxy)-phenyl]-imidazolidine-2,4-dione;-   (R)-3-[(S)-1-(5-Cyclopropyl-1H-benzoimidazol-2-yl)-2-phenyl-ethyl]-5-[4-(2-hydroxy-ethoxy)-phenyl]-imidazolidine-2,4-dione;-   (R)-3-[(S)-1-(5-Ethynyl-1H-benzoimidazol-2-yl)-2-phenyl-ethyl]-5-[4-(2-hydroxy-ethoxy)-phenyl]-imidazolidine-2,4-dione;    and-   (R)-3-[(1S,2S)-1-(5-Ethynyl-1H-benzoimidazol-2-yl)-2-phenyl-propyl]-5-[4-(2-hydroxy-ethoxy)-phenyl]-imidazolidine-2,4-dione;

or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the MEK inhibitor is a compound selected from thegroup consisting of GDC-0973 (Methanone,[3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl][3-hydroxy-3-(2S)-2-piperidinyl-1-azetidinyl]-),G-38963, G02443714, G02442104, and G00039805, or a pharmaceuticallyacceptable salt or solvate thereof.

IV Kits

In another aspect, provided is a kit comprising a PD-L axis bindingantagonist and/or a MEK inhibitor for treating or delaying progressionof a cancer in an individual or for enhancing immune function of anindividual having cancer. In some embodiments, the kit comprises a PD-1axis binding antagonist and a package insert comprising instructions forusing the PD-1 axis binding antagonist in combination with a MEKinhibitor to treat or delay progression of cancer in an individual or toenhance immune function of an individual having cancer. In someembodiments, the kit comprises a MEK inhibitor and a package insertcomprising instructions for using the MEK inhibitor in combination witha PD-1 axis binding antagonist to treat or delay progression of cancerin an individual or to enhance immune function of an individual havingcancer. In some embodiments, the kit comprises a PD-1 axis bindingantagonist and a MEK inhibitor, and a package insert comprisinginstructions for using the PD-1 axis binding antagonist and the MEKinhibitor to treat or delay progression of cancer in an individual or toenhance immune function of an individual having cancer. Any of the PD-1axis binding antagonists and/or MEK inhibitors described herein may beincluded in the kits.

In some embodiments, the kit comprises a container containing one ormore of the PD-1 axis binding antagonists and MEK inhibitors describedherein. Suitable containers include, for example, bottles, vials (e.g.,dual chamber vials), syringes (such as single or dual chamber syringes)and test tubes. The container may be formed from a variety of materialssuch as glass or plastic. In some embodiments, the kit may comprise alabel (e.g., on or associated with the container) or a package insert.The label or the package insert may indicate that the compound containedtherein may be useful or intended for treating or delaying progressionof cancer in an individual or for enhancing immune function of anindividual having cancer. The kit may further comprise other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, and syringes.

EXAMPLES

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

Example 1 MEK Inhibitor Enhanced MHC I Expression on Tumor Cell Lines

To determine if treatment with MEK inhibitor (MEKi) enhancedimmunogenicity of tumor cells, surface expression of MHC-I on tumor celllines treated with MEK inhibitors GDC-0973 and G-38963 was assayed.Briefly, human melanoma cell lines (Malme-3M, A2058, A375, HS294T, SK23,SKMEL-28, 537 MeI, RPMI-795) and human colorectal cell lines (Colo 320DM, Colo 205, WiDr, Colo 741, RKO, DLD-1, HM7, HCT-15) were treated with1 micromolar MEKi GDC-0973 or G-38963, BRAF inhibitor (BRAFi) GDC-0879,or DMSO vehicle for 24 hours. Following treatment, cells were stainedfor surface MHC Class I expression with an antibody against HLA-A,B,Cfor subsequent FACS analysis. Data shown is for treatment with MEKiGDC-0973. Labeled isotype-matched antibodies were used to determine thelevel of non-specific staining. Data analysis and construction ofhistograms demonstrated that cell surface expression of MHC-I wasupregulated in MEKi treated cells as compared to vehicle treated cells(FIG. 1A). In contrast, cell surface expression of MHC-I in BRAFitreated cells was not upregulated as compared to vehicle treated cells(FIG. 2). These results demonstrate that enhanced cell surfaceexpression of MHC-I in both melanoma and colorectal tumor cells isspecific to MEK inhibition and not due to general inhibition of theRAS/RAF/MEK signaling pathway.

To determine if treatment with MEK inhibitor (MEKi) enhancedimmunogenicity of mouse tumor cells similarly to human tumor cells,surface expression of MHC-I on mouse tumor cell lines treated with MEKiGDC-0973 was assayed. Briefly, mouse melanoma cell lines (MC38 andB16.F10) and a mouse colorectal cell line (CT26) were treated with MEKiGDC-0973, G-38963 or vehicle. Briefly, cells were stimulated for 24hours with 1 micromolar MEK inhibitor or DMSO vehicle control. Followingtreatment, cells were surfaced stained with an antibody against MHC-I(H-2D) and expression was assayed by subsequent FACS analysis. Labeledisotype-matched antibodies were used to determine the level ofnon-specific staining. Data analysis and construction of histogramsdemonstrated that cell surface expression of MHC-I was upregulated inMEKi treated cells (data shown is for MEKi GDC-0973) as compared tovehicle treated cells (FIG. 1B). These results demonstrate that enhancedcell surface expression of MHC-I occurred across several melanoma andcolorectal tumor cell lines regardless of mouse or human origin.

To determine if enhanced cell surface expression of MHC-I is specific totumor cells, the effect of MEKi treatment on MHC-I expression on humanperipheral blood mononuclear cells (PMBCs) was assayed. Briefly, PMBCSwere isolated from whole blood by first diluting it with an equal volumeof room temperature PBS and subsequent overlay onto Ficoll-filledLeucosep tubes (Greiner Bio-One). Post-centrifugation, the PBMCinterface was then washed twice and resuspended in culture media(RPMI-1640 with 10% fetal bovine serum, 20 μM HEPES, 55 μM2-mercaptoethanol, 50 μg/ml gentamicin, and 1:100 dilutions of thefollowing supplements from Gibco: Gluta-MAX, sodium pyruvate,penicillin/streptomycin, and non-essential amino acids). Cells wereplated in 6 well plates at 4×10⁶ per well with a total of 4 ml per well.MEK inhibitor GDC-0973 was added at either 1 μM or 3 μM. Cells wereharvested 24 hours later and distributed to a 96-well V-bottom plate forFACS staining. Cells were stained with the following antibodies (allfrom BD Biosciences, at 1:10 for 30 minutes on ice): CD3-FITC,HLA-ABC-PE, CD4-APC, CD19-FITC, and CD14-FITC. Propidium iodide wasincluded to exclude dead cells. Samples were run on a BD FACSCaliberflow cytometer and data was analyzed using FlowJo software (Tree Star,Inc.). Data analysis and construction of histograms demonstrated thatcell surface expression of MHC-I was not upregulated in CD4+ T cells(FIG. 3A), CD8+ T cells (FIG. 3B), B cells (FIG. 3C), or monocytes (FIG.3D) treated with 1 μM MEKi GDC-0973 or 3 μM MEKi GDC-0973 as compared tovehicle treated cells. These results demonstrate that enhanced cellsurface expression of MHC-I by MEK inhibitor treatment is specific totumor cells.

Example 2 Co-Stimulatory Signals Made T Cells Resistant to TCR SignalingInactivation by MEK Inhibitor

Recent studies have shown that MEK inhibitor treatment impairs Tlymphocyte function (Boni et al., Cancer Res., 70(13), 2010). To confirmthat MEK inhibitor treatment impaired CD8+ T cells, T cells were treatedwith MEKi in combination with T cell stimulation signals and assayed forT cell proliferation. Briefly, human CD8+ T cells were purified fromwhole blood using StemCell Technologies human CD8 RosetteSep as permanufacturer's instructions. Purified cells were plated at 200,000 perwell in triplicate in 96-well U-bottom plates with 200,000 per well ofeither anti-CD3 or anti-CD3/anti-CD28 Dynabeads (Invitrogen). MEKinhibitors GDC-0973 and G-38963 were titrated 10-fold from 10 μM to0.001 μM such that the final culture concentration was 0.5% DMSO in atotal volume of 200 μl per well. Culture medium was RPMI-1640 with 10%fetal bovine serum, 20 μM HEPES, 55 μM 2-mercaptoethanol, 50 μg/mlgentamicin, and 1:100 dilutions of the following supplements from Gibco:Gluta-MAX, sodium pyruvate, penicillin/streptomycin, and non-essentialamino acids. At 48 hours, wells were pulsed with 1 μCi/well of3H-thymidine and cultured an additional 16 hours prior to freezing andharvest. Data analysis demonstrated that treatment of CD8+ T cells withanti-CD3 stimulated T cell activation (closed triangle) as compared tounstimulated T cells (open circle). Treatment of T cells with twodifferent MEK inhibitors reduced the stimulatory effect of anti-CD3(closed circle, closed square) at all MEKi concentrations tested, withnearly complete inhibition of T cell receptor induced proliferationoccurring at 0.01 μM MEKi treatment (FIG. 4A). In contrast,co-stimulation with anti-CD3 and anti-CD28 in MEKi treated T cells(closed circle, closed square) was sufficient to overcome the inhibitoryeffect of MEKi on T cell activation (FIG. 4B). These unexpected resultsdemonstrate the novel finding that inhibition of TCR signaling by MEKitreatment can be overcome by providing sufficient T cell co-stimulationwhich is provided to T cells by antigen presenting cells such as Bcells, macrophages, and dendritic cells.

Without being bound to theory, a key component of co-stimulation isthought to be the activation of PI3 kinase and is provided by CD28 viaassociation of PI3K p85 subunit with its cytoplsmic YMNM motif. PD-1,through its interaction with SHP2, impedes the activity of PI3K.Therefore, blockade of the PDI axis may disinhibit PI3kinase, resultingin enhanced T cell costimulation and provides a means to overcome theinhibitory effect of MEKi on T cell activation. PD-1/-L1 blockade is toenhance co-stimulation under conditions when expression ofco-stimulatory ligands such as B7.1 and B7.2 is often limiting such asin most tumors or the tumor microenvironment. Combining MEKi withblockade of the PDI axis should enhance tumor specific T cell immunityby enhancing Ag recognition by the TCR through upregulation of tumor MHCI (enhancing Signal I) by MEKi and by relieving inhibition of PI3K(enhancing Signal 2) through PD1/PDL1 blockade.

Example 3 MEK Inhibitor Specifically Enhanced Maturation and Activationof Dendritic Cells

To determine if MEK inhibitor treatment specifically enhanced tumorimmunogenicity by stimulating dendritic cells (DCs), monocyte-deriveddendritic cells were treated with increasing concentration of MEKiGDC-0973, MEKi GDC-38963 or BRAFi GDC-0879 in combination withantibodies to the DC co-stimulatory molecule CD40. Briefly, humanmonocytes were purified from whole blood using StemCell Technologieshuman monocyte RosetteSep as per manufacturer's instructions. Monocyteswere seeded in T175 flasks at approximately 0.5-1.0×10⁶ per ml in 50ng/ml human GM-CSF and 100 ng/ml human IL-4 for 7 days total, withhalf-media exchanges every 2 days. Cells were then harvested and platedat 100,000 cells/well in 96-well flat bottom plates with or withoutPfizer anti-CD40 at 1 g/ml. MEK inhibitors and BRAF inhibitor weretitrated 10-fold from 10 M to 0.001p M such that the final cultureconcentration was 0.5% DMSO in a total volume of 200 μl per well.Forty-eight hours later, cells were harvested and transferred to a96-well V-bottom plate. Cells were first Fc-receptor blocked (Miltenyi)and then stained using the following antibodies (from BD Biosciences at1:10, 30 minutes on ice): HLA-DR,-DP,-DQ-FITC, HLA-ABC-PE, CD83-APC,CD14-FITC, CD80-PE, and CD86-APC. Propidium iodide was included toexclude dead cells. Samples were run on a BD FACSCaliber flow cytometerand data was analyzed using FlowJo software (Tree Star, Inc.). Dataanalysis and construction of histograms demonstrated that the frequencyof cells expressing the maturation marker CD83 (FIG. 5A), MHC-II (FIG.5B), and co-stimulatory molecule CD86 (FIG. 5C) was increased in cellstreated with 1 μM MEKi GDC-0973 as compared to vehicle treated cells. Incontrast, cell surface expression of these DC surface activation markersin DCs treated with 1 μM BRAFi was not upregulated and was similar tovehicle treated cells. Furthermore, increasing concentrations of eitherMEKi G-38963 (closed square) or MEKi GDC-0973 (closed circle) enhancedthe frequency of DCs expressing these surface markers of DC maturationand activation in concentration dependent manner (FIG. 5D-SF). Incontrast BRAFi (closed triangle) treatment did not enhance the anti-CD40co-stimulatory effect. These novel results demonstrate that enhancedmaturation and activation of DCs is specific to MEK inhibitor treatmentand not due to general inhibition of the RAS/RAF/MEK signaling pathway.Furthermore, MEKi enhanced activation of human monocyte-derived DCsco-stimulated with anti-CD40 in a concentration dependent mannerindicating that MEKi may have an immunomodulatory effect on DCs.

Example 4 Co-Treatment with MEK Inhibitor and Anti-PD-L1 AntibodiesReduced Serum Levels of Cytokines that Promote Tumor Growth

Due to the novel observation that MEKi treatment enhanced T cell and DCactivation in the presence of a co-stimulator, MEKi G-38963 was used incombination with anti-PD-L1 antibodies to determine if MEKi couldenhance the anti-tumor effects of anti-PD-L1 antibody treatment andmodulate cytokine levels in tumor bearing animals. The anti-PD-L1antibody employed in these experiments was PRO314483, LOT#59554.96,raised against human PD-L1 and recognizes both human and murine PD-L.Briefly, 7 days after treatment, mice were anaesthetized and bledretro-orbitally for serum. Analysis for serum levels of cytokines wasconducted using the BioRad Bio-Plex assay and it was determined that theimmunosuppressive cytokine IL-10 was significantly reduced in in vivomodels for both melanoma (FIG. 6A) and colorectal (FIG. 6C) tumors.IL-10 levels were decreased with anti-PD-L1 antibody or MEKi treatmentalone but were significantly reduced by co-treatment with MEKi andanti-PD-L1 antibodies. Furthermore, serum levels of the murine chemokineKC, homolog of the human chemokine IL-8 that is known to play a role intumor progression, was also significantly reduced in in vivo models forboth melanoma (FIG. 6B) and colorectal (FIG. 6D) tumors with the mostsignificant reduction induced by co-treatment with MEKi and anti-PD-L1antibodies. These results indicate that combination treatment ofanti-PD-L1 antibodies and MEKi inhibits release of cytokines thatpromote tumor growth.

Example 5 MEK Inhibition Enhanced Anti-Tumor Activity of Anti-PD-L1Antibodies in Colorectal Tumors In Vivo

To determine if MEKi enhanced the anti-tumor effect of anti-PD-L1antibodies, mouse models for colorectal tumors were treated with thecombination treatment. Briefly, mice were inoculated subcutaneously withtumor cells and allowed to grow tumors. When tumor bearing mice achieveda mean tumor volume of 200 mm3 (FIG. 7A) or 450 mm3 (FIG. 7B), mice wererandomly assigned to I of 4 treatment groups. Group 1: received 10 mg/kgof an isotype control antibody (anti-gp120, PRO67181, PUR#20455)intraperitoneally three times a week for 3 weeks plus MCT controlvehicle, orally, daily for 21 days; Group 2: received 10 mg/kganti-PD-L1 antibody PRO314483, LOT#59554.96 intraperitoneally threetimes a week for three weeks; Group 3: received 10 mg/kg of an isotypecontrol antibody (anti-gp120, PRO67181, PUR#20455) intraperitoneally3×/week×3 plus 75 mg/kg MEKi G-38963, orally, daily for 21 days; Group4: received 10 mg/kg of an anti-PD-L1 antibody PRO314483, LOT#59554.96intraperitoneally three times a week for three weeks plus 75 mg/kg MEKiG-38963, orally, daily for 21 days. Mice were monitored for tumor growthand body weight changes. Blockade of PD-L1 with anti-PD-L1 antibodyPRO314483, LOT#5944.96 either in early (FIG. 7A) or in late (FIG. 7B)intervention was highly effective as a single agent therapy atpreventing tumor growth. Treatment with MEKi G-38963 was also highlyeffective as a single agent therapy at preventing tumor growth either inearly or in late intervention and was comparable to anti-PD-L1 antibodytreatment. Combination treatment with anti-PD-L1 antibodies and MEKisignificantly inhibited tumor growth both in early and late interventionand was significantly more effective than anti-PD-L1 antibodies or MEKitreatment alone. Furthermore, co-treatment at an early stage of tumorgrowth resulted not only in significant reduction of tumor volume butalso demonstrated a sustained response. Early intervention resulted inabout a 60% complete response that was maintained for at least 92 days.These results indicate that MEKi enhanced the anti-tumor activity ofPD-L1 blockade and therefore worked synergistically with anti-PD-L1antibodies to inhibit tumor growth.

To further determine if MEKi enhanced the anti-tumor effect ofanti-PD-L1 antibodies, mouse models for colorectal tumors were treatedwith the combination treatment using a different MEK inhibitor, MEKiGDC-0973, in two different studies.

For the first study, female BALB/c mice were inoculated subcutaneouslyin the unilateral thoracic region with 100,000 CT26 murine colorectalcells in 100 μL of HBSS:matrigel. When mice achieved a mean tumor volumeof approximately 200 mm3, they were randomly assigned to one of ninedifferent treatment groups on experimental day 0 and treatment wasinitiated on experimental day I. Groups of 10 mice were orally given thefollowing in a volume of 200 μl daily for 21 days: Group 1 received MCTvehicle; Group 2 received 0.5 mg/kg GDC-0973; Group 3 received 1.0 mg/kgGDC-0973; Group 4 received 2.0 mg/kg GDC-0973; Group 5 received 3.0mg/kg GDC-0973; Group 6 received 4.0 mg/kg GDC-0973; Group 7 received5.0 mg/kg GDC-0973; Group 8 received 6.0 mg/kg GDC-0973; and Group 9received 7.5 mg/kg GDC-0973.

For the second study, female BALB/c mice were inoculated subcutaneouslyin the unilateral thoracic region with 100,000 CT26 murine colorectalcells in 100 μL of HBSS:matrigel. When mice achieved a mean tumor volumeof approximately 200 mm3, they were randomly assigned to one of sixdifferent treatment groups on experimental day 0 and treatment wasinitiated on experimental day 1. Groups of 10 mice were given thefollowing: Group 1 received MCT vehicle orally in 200 uL volume dailyfor 21 days and 10 mg/kg of an isotype control antibody (anti-gp120,PRO67181, PUR#20455) intraperitoneally 3 times per week; Group 2received 7.5 mg/kg GDC-0973 orally daily for 21 days; Group 3 received10 mg/kg anti-PD-L1 antibody PRO314483, LOT#5944.96 intraperitoneally 3times per week; Group 4 received 10 mg/kg anti-PD-L1 antibody*PRO314483,LOT#5944.96 intraperitoneally 3 times per week and 1.0 mg/kg GDC-0973orally daily for 21 days; Group 5 received 10 mg/kg anti-PD-L1 antibodyPRO314483, LOT#5944.96 intraperitoneally 3 times per week and 3.0 mg/kgGDC-0973 orally daily for 21 days; and Group 6 received 10 mg/kganti-PD-L1 antibody PRO314483, LOT#5944.96 intraperitoneally 3 times perweek and 6.0 mg/kg GDC-0973 orally daily for 21 days. The anti-PD-L1antibody PRO314483, LOT#5944.96 was a reverse chimera, containing thehuman variable region of MPDL3280A and the murine constant region ofIgG2A, with an effector-less Fc D265A/N297A substitution in the constantregion.

For both studies, mice were monitored for tumor growth and body weightchanges two to three times per week for the duration of the study. Formeasurement of tumor growth, tumor volume was measured using UltraCal-IVcalipers (Model 54-10-11; Fred V. Fowler Company; Newton, Mass.) withlength and width measurements perpendicular to one another, and tumorvolume was calculated using the equation:

Tumor Volume(mm³)=(Length×Width²)×0.5

For measurement of body weights, mice were weighed using an AdventuraPro AV812 scale (Ohaus Corporation; Pine Brook, N.J.). Percent bodyweight change was calculated using the equation:

Body weight change(%)=[(Weight_(Day new)−Weight_(Day 0))/Weight_(Day 0)]×100

Data was analyzed using R, version 2.9.2 (R Development Core Team 2008;R Foundation for Statistical Computing; Vienna, Austria), and the mixedmodels were fit within R using the nlme package, version 3.1-96(Pinheiro J et al., R package version 3. 2009, 1-96). Plotting wasperformed in Prism, version 5.0b for Mac (GraphPad Software, Inc.; LaJolla, Calif.). A mixed modeling approach was used to analyze therepeated measurement of tumor volumes from the same animals over time(Pinheiro J et al., Statistics and Computing, Springer. 2010). Thisapproach addressed both repeated measurements and modest dropouts beforestudy end for reasons classifiable statistically as missing at random(MAR). The fixed effect changes in log₂ (volume) by time and dose aremodeled as the sum of the main effects and interaction of a naturalcubic regression spline basis in time with an auto-determined naturalspline basis in dose. Intercepts and growth rates (slopes) were assumedto vary randomly by animal. Tumor growth inhibition as a percentage ofthe control-treated group (% TGI) was calculated as the percentage ofthe area under the fitted curve (AUC) for the respective treatment groupper day in relation to the control while the control treated mice werestill on study, using the equation:

% TGI=100×(1−AUC _(dose) /AUC _(vehicles))

Complete Response (CR) was defined as an individual animal whose tumorvolume fell below the Limit of Detection (LOD), at any time during thestudy. Partial Response (PR) was defined as an individual animal whosetumor volume decreased by 50% of its initial tumor volume at any timeduring the study. Overall Response Rate (ORR) was defined as the sum ofthe complete and partial responses.

Time To Progression 5X (TTP5X) was defined as the time in days for agroup's fitted tumor volume (based upon the mixed modeling analysisdescribed above) to exceed 5 times the starting volume, rounded to thenearest half day and reported as the TTP5X for that group. Linearmixed-effects analysis was also employed to analyze the repeatedmeasurement of body weight changes from the same animals over time.

Treatment with increasing concentrations of MEKi GDC-0973 suppressedtumor growth with maximal inhibition demonstrated by the 7.5 mg/kgGDC-0973 treatment group at 20 days post-treatment (FIG. 8A, Table 2).

TABLE 2 Increased TGI due to increasing doses of MEKi GDC-0973 Treatment% TGI Vehicle 0 GDC-0973, 0.5 mg/kg −8 GDC-0973, 1.0 mg/kg −16 GDC-0973,2.0 mg/kg −21 GDC-0973, 3.0 mg/kg −4 GDC-0973, 4.0 mg/kg 27 GDC-0973,5.0 mg/kg 55 GDC-0973, 6.0 mg/kg 72 GDC-0973, 7.5 mg/kg 87

Combination treatment with the anti-PD-L1 antibody and MEKi GDC-0973demonstrated enhanced reduction of tumor growth for a longer period oftime as compared to treatment with anti-PD-L1 antibodies or MEKiGDC-0973 alone (FIG. 8B, Table 3). Furthermore, lower dosageconcentrations of MEKi GDC-0973 (1 mg/kg, 3 mg/kg, and 6 mg/kg) weremore effective at suppressing tumor growth when used in combination withthe anti-PD-L1 antibody as compared to when a higher dosageconcentration of MEKi GDC-0973 was used alone (7.5 mg/kg) (FIGS. 8A andB, Table 3).

TABLE 3 Effectiveness of anti-PD-L1 antibody and MEKi GDC-0973combination treatment TTP5X Treatment %TGI (days) % PR % CR Control 0 120 0 anti-PD-L1 antibody 78 24 20 0 GDC-0973, 7.5 mg/kg 71 21.5 10 0anti-PD-L1 antibody + 78 30 20 10 GDC-0973, 1.0 mg/kg anti-PD-L1antibody + 98 43 30 20 GDC-0973, 3.0 mg/kg anti-PD-L1 antibody + 10644.5 40 20 GDC-0973, 6.0 mg/kg

Further studies were conducted to determine if additional MEK inhibitors(G02443714, G02442104, and G00039805) also enhanced the anti-tumoreffect of anti-PD-L1 antibodies when used for combination treatment in amouse model for colorectal tumors.

For combination treatment with the MEK inhibitor G02443714, femaleBALB/c mice were inoculated subcutaneously in the unilateral thoracicregion with 100,000 CT26 murine colorectal cells in 100 μL ofHBSS:matrigel. When mice achieved a mean tumor volume of approximately200 mm³, they were randomly assigned to one of four different treatmentgroups on experimental day 0 and treatment was initiated on experimentalday 1. Groups of 10 mice were given the following: Group 1 received MCTvehicle orally in 200 uL volume daily for 21 days and 10 mg/kg of anisotype control antibody (anti-gp120, PRO67181, PUR#20455)intraperitoneally 3 times per week; Group 2 received 25 mg/kg G02443714orally daily for 21 days; Group 3 received 10 mg/kg anti-PD-L1 antibodyPRO314483, LOT#5944.96 intraperitoneally 3 times per week; and Group 4received 10 mg/kg anti-PD-L1 antibody PRO314483, LOT#5944.96intraperitoneally 3 times per week and 25 mg/kg G02443714 orally dailyfor 21 days. G02443714 as well as oral vehicle (MCT) were dosed orallyby gavage four hours prior to administration of anti-PD-L1 and/orisotype control antibody.

For combination treatment with the MEK inhibitor G02442104, femaleBALB/c mice were inoculated subcutaneously in the unilateral thoracicregion with 100,000 CT26 murine colorectal cells in 100 μL ofHBSS:matrigel. When mice achieved a mean tumor volume of approximately200 mm³, they were randomly assigned to one of four different treatmentgroups on experimental day 0 and treatment was initiated on experimentalday 1. Groups of 10 mice were given the following: Group 1 received MCTvehicle orally in 200 uL volume daily for 21 days and 10 mg/kg of anisotype control antibody (anti-gp120, PRO67181, PUR#20455)intraperitoneally 3 times per week; Group 2 received 25 mg/kg G02442104orally daily for 21 days; Group 3 received 10 mg/kg anti-PD-L1 antibodyPRO314483, LOT#5944.96 intraperitoneally 3 times per week; and Group 4received 10 mg/kg anti-PD-L1 antibody PRO314483, LOT#5944.96intrapeiitoneally 3 times per week and 25 mg/kg G02442104 orally dailyfor 21 days. G02442104 as well as oral vehicle (MCT) were dosed orallyby gavage four hours prior to administration of anti-PD-L1 and/orisotype control antibody.

For combination treatment with the MEK inhibitor G00039805, femaleBALB/c mice were inoculated subcutaneously in the unilateral thoracicregion with 100,000 CT26 murine colorectal cells in 100 μL ofHBSS:matrigel. When mice achieved a mean tumor volume of approximately200 mm3, they were randomly assigned to one of four different treatmentgroups on experimental day 0 and treatment was initiated on experimentalday 1. Groups of 10 mice were given the following: Group 1 received MCTvehicle orally in 200 uL volume daily for 21 days and 10 mg/kg of anisotype control antibody (anti-gp120, PRO67181, PUR#20455)intraperitoneally 3 times per week; Group 2 received 100 mg/kg G00039805orally daily for 21 days; Group 3 received 10 mg/kg anti-PD-L1 antibodyPRO314483, LOT#5944.96 intraperitoneally 3 times per week; and Group 4received 10 mg/kg anti-PD-L1 antibody PRO314483, LOT#5944.96intraperitoneally 3 times per week and 100 mg/kg G00039805 orally dailyfor 21 days. G00039805 as well as oral vehicle (MCT) were dosed orallyby gavage four hours prior to administration of anti-PD-L1 and/orisotype control antibody.

For all three combination studies with G02443714, G02442104, orG00039805, mice were monitored for tumor growth and body weight changestwo to three times per week for the duration of the study. Formeasurement of tumor growth, tumor volume was measured using UltraCal-IVcalipers (Model 54-10-111; Fred V. Fowler Company; Newton, Mass.) withlength and width measurements perpendicular to one another, and tumorvolume was calculated using the equation:

Tumor Volume (mm³)=(Length×Width²)×0.5

For measurement of body weights, mice were weighed using an AdventuraPro AV812 scale (Ohaus Corporation; Pine Brook, N.J.). Percent bodyweight change was calculated using the equation:

Body weight change(%)=[(Weight_(Day new)−Weight_(Day 0))/Weight_(Day 0)]×100

Data was analyzed using R, version 2.9.2 (R Development Core Team 2008;R Foundation for Statistical Computing; Vienna, Austria), and the mixedmodels were fit within R using the nlme package, version 3.1-96(Pinheiro J et al., R package version 3. 2009, 1-96). Plotting wasperformed in Prism, version 5.0b for Mac (GraphPad Software, Inc.; LaJolla, Calif.). A mixed modeling approach was used to analyze therepeated measurement of tumor volumes from the same animals over time(Pinheiro J et al., Statistics and Computing, Springer. 2010). Thisapproach addressed both repeated measurements and modest dropouts beforestudy end for reasons classifiable statistically as missing at random(MAR). The fixed effect changes in log₂ (volume) by time and dose aremodeled as the sum of the main effects and interaction of a naturalcubic regression spline basis in time with an auto-determined naturalspline basis in dose. Intercepts and growth rates (slopes) were assumedto vary randomly by animal. Tumor growth inhibition as a percentage ofthe control-treated group (% TGI) was calculated as the percentage ofthe area under the fitted curve (AUC) for the respective treatment groupper day in relation to the control while the control treated mice werestill on study, using the equation:

% TGI=100×(1−AUC _(dose)/AUC_(vehicles))

Complete Response (CR) was defined as an individual animal whose tumorvolume fell below the Limit of Detection (LOD), at any time during thestudy. Partial Response (PR) was defined as an individual animal whosetumor volume decreased by 50% of its initial tumor volume at any timeduring the study. Overall Response Rate (ORR) was defined as the sum ofthe complete and partial responses.

Time To Progression 5X (TTP5X) was defined as the time in days for agroup's fitted tumor volume (based upon the mixed modeling analysisdescribed above) to exceed 5 times the starting volume, rounded to thenearest half day and reported as the TTP5X for that group. Linearmixed-effects analysis was also employed to analyze the repeatedmeasurement of body weight changes from the same animals over time.

Combination treatment with the anti-PD-L1 antibody and G02443714resulted in enhanced reduction of tumor growth for a longer period oftime as compared to treatment with anti-PD-L1 antibodies or G02443714alone with a 20% partial response observed at 18 days (FIG. 9).Combination treatment with the anti-PD-L1 antibody and G02442104 alsoresulted in enhanced reduction of tumor growth for a longer period oftime as compared to treatment with anti-PD-L antibodies or MEKiG02442104 alone with a 40% partial response and 10% complete responseobserved at 37.5 days (FIG. 10). In addition, combination treatment withthe anti-PD-L1 antibody and G00039805 resulted in enhanced reduction oftumor growth for a longer period of time as compared to treatment withanti-PD-L1 antibodies or MEKi GOQ039805 alone with a 30% partialresponse observed at 22 days (FIG. 1). Altogether these resultsdemonstrate that a variety of MEK inhibitors can enhance the anti-tumoractivity of anti-PD-L1 antibodies to inhibit tumor growth.

Example 6 MEK Inhibition Enhanced Anti-Tumor Activity of Anti-PD-L1Antibodies in Melanoma Tumors In Vivo

To determine if MEKi enhanced the anti-tumor effect of anti-PD-L1antibodies, mouse models for melanoma tumors were treated with thecombination treatment. Briefly, mice were inoculated subcutaneously withtumor cells and allowed to grow tumors. When tumor bearing mice achieveda mean tumor volume of 100-200 mm3, mice were randomly assigned to 1 of4 treatment groups. Group 1: received 10 mg/kg of an isotype controlantibody (anti-gp120, PRO67181, PUR#20455) intraperitoneally three timesa week for three weeks plus MCT control vehicle, orally, daily for 21days; Group 2: received 10 mg/kg anti-PD-L1 antibody PRO314483,LOT#59554.96 intraperitoneally three times a week for three weeks; Group3: received 10 mg/kg of an isotype control antibody (anti-gp120,PRO67181, PUR#20455) intraperitoneally three times a week for threeweeks plus 75 mg/kg MEKi G-38963, orally, daily for 21 days; Group 4:received 10 mg/kg of an anti-PD-L1 antibody PRO314483, LOT#59554.96intraperitoneally three times a week for three weeks plus 75 mg/kg MEKiG-38963, orally, daily for 21 days. Mice were monitored for tumor growthand body weight changes. Blockade of PD-L1 with anti-PD-L1 antibodyPRO314483, LOT#59554.96 in Cloudman S91 (FIG. 12) melanoma tumors waseffective as a single agent therapy at preventing tumor growth.Treatment with MEKi G-38963 was also highly effective as a single agenttherapy at preventing tumor growth (FIG. 12) and was comparable toanti-PD-L1 antibody treatment. Combination treatment with anti-PD-L1antibodies and MEKi significantly inhibited tumor growth in bothmelanoma cell lines. In contrast, Temodar, a chemotherapeutic agent,when used in combination with anti-PD-L1 antibodies inhibited theanti-tumor activity of anti-PD-L 1 antibodies (FIG. 13). Similar resultswere obtained when an antibody that blocks the T cell OX40co-stimulatory molecule was used in combination with the MEK inhibitorG-38963 (FIG. 14). These results indicate that MEKi specificallyenhanced the anti-tumor activity of PD-L1 blockade and therefore workedsynergistically with anti-PD-L1 antibodies to inhibit melanoma tumorgrowth.

Example 7 MEK Inhibitor Increased Activation of Dendritic CellsIndependently of PDL1 Antibody Activity

Previous studies have indicated that MEK inhibition can augment immunefunction by downregulation of surface PD-L1 suggesting that the effectsof MEKi were mediated via alterations in PD-L1 expression. To determineif enhanced tumor immunogenicity is due to dependency of PD-L Iexpression upon MEK activation, activation of dendritic cells wascompared when treated with MEKi GDC-0973 alone, anti-PD-L1 antibodies (achimeric antibody composed of variable regions of MPDL3280A fused tomouse IgG2a constant sequences that contain an Fc mutation to preventeffective binding to Fcgamma receptors) alone or MEKi in combinationwith anti-PD-L1 antibodies. Briefly, mouse bone marrow cells wereisolated and seeded at 2×10⁶ per 10 ml total volume per 10 cm non-tissueculture treated dishes with 40 ng/ml mouse GM-CSF for 7 days. Freshmedia was half-exchanged every 2-3 days. Culture medium was RPMI-1640with 10% fetal bovine serum, 20 μM HEPES, 55 μM 2-mercaptoethanol, 50μg/ml gentamicin, and 1:100 dilutions of the following supplements fromGibco: Gluta-MAX, sodium pyruvate, penicillin/streptomycin, andnon-essential amino acids. On day 7 all cells were harvested and washed,then seeded at 100,000 cells/well in a 96-well flat-bottom plate. MEKinhibitor GDC-0973 was added at a final concentration of 1 μM, anti-PDLIhuman/mouse reverse chimera or anti-Ragweed mouse IgG2a isotype control(Genentech PUR 22251) were added at 10 μg/ml. Prior to adding to cellsfor a final concentration of 1 μg/ml each, anti-CD40 clone FGK-45(Genentech lot 68020-62) was crossed-linked with goat anti-Rat IgGFc-gamma-receptor (Jackson ImmunoResearch) at room temperature for onehour. After 48 hours of stimulation, cells were harvested andtransferred to a 96-well V-bottom plate. Samples were first Fc receptorblocked (purified anti-CD16/CD32 from BD Biosciences, 5 μg/ml) and thenstained with I-A/I-E-FITC, H-2 Db/H-2 Kb-biotin (followed bystreptavidin-PE), CD11c-APC, CD86-FITC, and CD80-PE (all from BDBiosciences). Propidium iodide was included to exclude dead cells.Samples were run on a BD FACSCaliber flow cytometer and data wasanalyzed using FlowJo software (Tree Star, Inc.). Treatment withfunctionally blocking anti-PD-L1 antibodies alone modestly increased DCsurface expression of MHC-I (FIG. 15A) however it did not induceexpression of DC surface activation markers MHC-II (FIG. 15B), CD80(FIG. 15C), or CD86 (FIG. 15D). In contrast MEKi treatment enhancedMHC-II, CD80, and CD86 as well as MHC-I expression. Interestingly,combination treatment of MEKi and anti-PD-L1 antibodies did not alter DCsurface activation markers as compared to MEKi alone. Similar resultswere obtained with the addition of the co-stimulatory anti-CD40antibodies (FIG. 15E-H). These novel findings indicate that MEKi inducedactivation of DCs independently of its effect on PD-L expression.Altogether these results demonstrate that MEKi increased tumorimmunogenicity by mechanisms unique from anti-PDL and provide supportfor combining MEKi and PD-L blockade for optimal enhancement ofanti-tumor immunity.

Example 8a MEK Assay (MEK Activity Assay)

Constitutively activated human mutant MEK1 expressed in insect cells isused as source of enzymatic activity at a final concentration in thekinase assay of 62.5 nM.

The assay is carried out for 30 minutes in the presence of 50 μM ATPusing recombinant GST-ERKl produced in E. Coli as substrate.Phosphorylation of the substrate is detected and quantified using HTRFreagents supplied by Cisbio. These consist of an anti-GST antibodyconjugated to allophycocyanin (XL665) and an anti-phospho(Thr202/Tyr204) ERK antibody conjugated to europium-cryptate. Theanti-phospho antibody recognises ERK I dually phosphorylated on Thr202and Tyr204. When both antibodies are bound to ERK1 (i.e. when thesubstrate is phosphorylated), energy transfer from the cryptate to theallophycocyanin occurs following excitation at 340 nm, resulting influorescence being emitted that is proportional to the amount ofphosphorylated substrate produced. Fluorescence is detected using amultiwell fluorimeter.

Compounds are diluted in DMSO prior to addition to assay buffer and thefinal DMSO concentration in the assay is 1%.

The IC₅₀ is defined as the concentration at which a given compoundachieves 50% inhibition of control. IC₅₀ values are calculated using theXLfit software package (version 2.0.5).

Example 8b MEK Assay (MEK Activity Assay)

Constitutively activated human mutant MEK1 expressed in insect cells isused as source of enzymatic activity at a final concentration in thekinase assay of 15 nM.

The assay is carried out for 30 minutes in the presence of 50 μM ATPusing recombinant GST-ERK1 produced in E. Coli as substrate.Phosphorylation of the substrate is detected and quantified using HTRFreagents supplied by Cisbio. These consist of an anti-GST antibodyconjugated to allophycocyanin (XL665) and an anti-phospho(Thr202/Tyr204) ERK antibody conjugated to europium-cryptate. These areused at a final concentration of 4 μg/ml and 0.84 μg/ml respectively.The anti-phospho antibody recognises ERK1 dually phosphorylated onThr202 and Tyr204. When both antibodies are bound to ERK1 (i.e. when thesubstrate is phosphorylated), energy transfer from the cryptate to theallophycocyanin occurs following excitation at 340 nm, resulting influorescence being emitted that is proportional to the amount ofphosphorylated substrate produced. Fluorescence is detected using amultiwell fluorimeter.

Compounds are diluted in DMSO prior to addition to assay buffer and thefinal DMSO concentration in the assay is 1%.

The ICs, is defined as the concentration at which a given compoundachieves 50% inhibition of control. ICo₅ values are calculated using theXLfit software package (version 2.0.5).

All patents, patent applications, documents, and articles cited hereinare herein incorporated by reference in their entireties.

1. A method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and a MEK inhibitor.
 2. The method of claim 1, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.
 3. The method of claim 2, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.
 4. The method of claim 3, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners.
 5. The method of claim 4, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1.
 6. The method of claim 4, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2.
 7. The method of claim 4, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.
 8. The method of claim 4, wherein the PD-1 binding antagonist is an antibody.
 9. The method of claim 8, wherein the PD-1 binding antagonist is MDX-1106.
 10. The method of claim 8, wherein the PD-1 binding antagonist is Merck
 3745. 11. The method of claim 8, wherein the PD-1 binding antagonist is CT-011.
 12. The method of claim 4, wherein the PD-1 binding antagonist is AMP-224.
 13. The method of claim 2, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.
 14. The method of claim 13, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.
 15. The method of claim 13, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1.
 16. The method of claim 13, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.
 17. The method of claim 13, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.
 18. The method of claim 17, wherein the PD-L1 binding antagonist is selected from the group consisting of: YW243.55.S70, MPDL3280A, and MDX-1105.
 19. The method of claim 17, wherein the antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:15, HVR-H2 sequence of SEQ ID NO:16, and HVR-H3 sequence of SEQ ID NO:3; and a light chain comprising HVR-L1 sequence of SEQ ID NO:17, HVR-L2 sequence of SEQ ID NO:18, and HVR-L3 sequence of SEQ ID NO:19.
 20. The method of claim 17, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:21.
 21. The method of claim 2, wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.
 22. The method of claim 21, wherein the PD-L2 binding antagonist is an antibody.
 23. The method of claim 21, wherein the PD-L2 binding antagonist is an immunoadhesin.
 24. The method of claim 1, wherein the MEK inhibitor is a competitive inhibitor of MEK.
 25. The method of claim 1, wherein the MEK inhibitor is more selective against an activating KRAS mutation.
 26. The method of claim 1, wherein the MEK inhibitor is an allosteric inhibitor of MEK.
 27. The method of claim 1, wherein the MEK inhibitor is more selective against an activating BRAF mutation.
 28. The method of claim 1, wherein the MEK inhibitor is a compound of the formula (I), (II), (III), (IV), (V), (VI) or (VII), or a pharmaceutically acceptable salt or solvate thereof.
 29. The method of claim 1, wherein the MEK inhibitor is selected from the group consisting of G02442104, 0-38963, G02443714, G00039805 and GDC-0973, or a pharmaceutically acceptable salt or solvate thereof.
 30. The method of claim 1, wherein the MEK inhibitor is G02443714, G02442104 or G00039805.
 31. The method of claim 1, wherein the cancer contains a BRAF V600E mutation.
 32. The method of claim 1, wherein the cancer contains a BRAF wildtype.
 33. The method of claim 1, wherein the cancer contains a KRAS wildtype.
 34. The method of claim 1, wherein the cancer contains an activating KRAS mutation.
 35. The method of claim 1, wherein the treatment results in a sustained response in the individual after cessation of the treatment.
 36. The method of claim 1, wherein the MEK inhibitor is administered continuously.
 37. The method of claim 1, wherein the MEK inhibitor is administered intermittently.
 38. The method of claim 1, wherein the MEK inhibitor is administered before the PD-1 axis binding antagonist.
 39. The method of claim 1, wherein the MEK inhibitor is administered simultaneous with the PD-1 axis binding antagonist.
 40. The method of claim 1, wherein the MEK inhibitor is administered after the PD-1 axis binding antagonist.
 41. The method of claim 1, wherein the individual has colorectal cancer.
 42. The method of claim 1, wherein the individual has melanoma.
 43. The method of claim 1, wherein the individual has non-small cell lung cancer.
 44. The method of claim 1, wherein the individual has ovarian cancer.
 45. The method of claim 1, wherein the individual has breast cancer.
 46. The method of claim 1, wherein the individual has pancreatic cancer.
 47. The method of claim 1, wherein the individual has a hematological malignancy.
 48. The method of claim 1, wherein the individual has renal cell carcinoma.
 49. A method of enhancing immune function in an individual having cancer comprising administering an effective amount of a combination of a PD-1 axis binding antagonist and a MEK inhibitor.
 50. The method of claim 49, wherein CD8 T cells in the individual have enhanced priming, activation, proliferation and/or cytolytic activity relative to prior to the administration of the combination.
 51. The method of claim 50, wherein the CD8 T cell activation is characterized by an elevated frequency of γ-IFN⁺ CD8 T cells and/or enhanced cytolytic activity relative to prior to administration of the combination.
 52. The method of claim 50, wherein the number of CD8 T cells is elevated relative to prior to administration of the combination.
 53. The method of claim 50, wherein the CD8 T cell is an antigen-specific CD8 T cell.
 54. The method of claim 50, wherein the cancer cells in the individual selectively have elevated expression of MHC class I antigen expression relative to prior to the administration of the PD-1 axis binding antagonist and the MEK inhibitor.
 55. The method of claim 54, wherein PBMC cells of the individual do not have elevated expression of MHC class I antigen.
 56. The method of claim 49, wherein the antigen presenting cells in the individual have enhanced maturation and activation relative prior to the administration of the PD-1 axis binding antagonist and the MEK inhibitor.
 57. The method of claim 56, wherein the antigen presenting cells are dendritic cells.
 58. The method of claim 56, wherein the maturation of the antigen presenting cells is characterized by increased frequency of CD83′ dendritic cells.
 59. The method of claim 56, wherein the activation of the antigen presenting cells is characterized by elevated expression of CD80 and CD86 on dendritic cells.
 60. The method of claim 50, wherein the serum levels of IL-10 and/or IL-8 in the individual are reduced relative to prior to the administration of the combination.
 61. The method of claim 50, wherein the cancer has elevated levels of T-cell infiltration.
 62. The method of claim 49, wherein the MEK inhibitor is a compound of the formula (I), (II), (III), (IV), (V), (VI) or (VII), or a pharmaceutically acceptable salt or solvate thereof.
 63. The method of claim 49, wherein the MEK inhibitor is selected from the group consisting of G02442104, G-38963, G02443714, G00039805 and GDC-0973, or a pharmaceutically acceptable salt or solvate thereof.
 64. The method of claim 49, wherein the PD-1 axis binding antagonist is an anti-PD-L1 antibody.
 65. The method of claim 64, wherein PD-L1 on the cancer cell surface is inhibited from transducing a signal to the intracellular pathway.
 66. The method of claim 64, wherein the anti-PD-L1 antibody is capable of inhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1.
 67. The method of claim 17, wherein the anti-PD-L1 antibody is a monoclonal antibody.
 68. The method of claim 17, wherein the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab′)₂ fragments.
 69. The method of claim 17, wherein the anti-PD-L1 antibody is a humanized antibody.
 70. The method of claim 17, wherein the anti-PD-L1 antibody is a human antibody.
 71. The method of claim 64, wherein the antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:15, HVR-H2 sequence of SEQ ID NO:16, and HVR-H3 sequence of SEQ ID NO:3; and a light chain comprising HVR-L1 sequence of SEQ ID NO:17, HVR-L2 sequence of SEQ ID NO:18, and HVR-L3 sequence of SEQ ID NO:19.
 72. The method of claim 64, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:21.
 73. The method of claim 1, wherein the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
 74. A kit comprising a PD-1 axis binding antagonist and a package insert comprising instructions for using the PD-1 axis binding antagonist in combination with a MEK inhibitor to treat or delay progression of cancer in an individual.
 75. A kit comprising a PD-1 axis binding antagonist and a MEK inhibitor, and a package insert comprising instructions for using the PD-1 axis binding antagonist and the MEK inhibitor to treat or delay progression of cancer in an individual.
 76. A kit comprising a MEK inhibitor and a package insert comprising instructions for using the MEK inhibitor in combination with a PD-1 axis binding antagonist to treat or delay progression of cancer in an individual. 77-79. (canceled) 